Therapeutic compounds comprised of anti-FC receptor binding agents

ABSTRACT

Multispecific molecules which target immune cells are disclosed. The molecules are “multispecific” because they bind to multiple (two or more), distinct targets, one of which is a molecule on the surface of an immune cell. Multispecific molecules of the invention include molecules comprised of at least one portion which binds to a molecule on an effector cell, such as an Fc receptor, and at least one portion (e.g., two, three, four or more portions) which binds to a different target, such as an antigen on a tumor cell or a pathogen. Multispecific molecules of the invention also include antigen “multimer complexes” comprised of multiple (i.e., two or more) portions which bind to a molecule on an antigen presenting cell (APC), such as an Fc receptor, linked to one or more antigens. These multimer complexes target antigens, such as self-antigens, to APCs to induce and/or enhance internalization (endocytosis), processing and/or presentation of the antigen by the APC. Therefore, these molecules can be used to induce or enhance an immune response either in vivo or in vitro against a normally non-immunogenic protein, such as a self-antigen.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 09/188,082,filed on Nov. 6, 1998, now U.S. Pat. No. 6,270,765, which is acontinuation of U.S. Ser. No. 08/661,052 filed on Jun. 7, 1996, nowissued as U.S. Pat. No. 5,837,243, which is a continuation-in-part ofSer. No. 08/484,172, entitled “Therapeutic Compounds Comprised ofAnti-Fc Receptor Antibodies” filed Jun. 7, 1995. The entire contents ofeach of the aforementioned applications and all references, issuedpatents, and published patent applications cited therein areincorporated herein by reference.

BACKGROUND OF THE INVENTION

Immunoglobulins (Igs) are composed of two heavy and two light chains,each of which contains an NH₂-terminal antigen-binding variable domainand a COOH-terminal constant domain responsible for the effectorfunctions of antibodies. The COOH-terminal domains of Ig heavy chainsform the Fc region and are involved in triggering cellular activitiesthrough interaction with specific receptors known as Fc receptors(FcRs). Fc receptors for all Ig classes, or isotypes, (e.g., IgG (FcγR),IgE (FcεR), IgA (FcαR), IgM (FcμR) and IgD (FcδR) have been identified.The different biological activities of antibodies of different isotypesare based in part on their ability to bind to different FcR expressed ondifferent immune (effector) cells (Fridman, W. H. (September 1991) TheFASEB Journal Vol. 5. 2684-2690). Murine antibodies, which are directedagainst FcRs have been made (See e.g. U.S. Pat. No. 4,954,617 entitledMonoclonal Antibodies To Fc Receptors for Immunoglobulin G on HumanMononuclear Phagocytes and International Patent Application PublicationNo. WO 91/05871 entitled Monoclonal Antibody Specific For IgA Receptor).

Murine monoclonal antibodies can be useful as human therapeutics and canbe produced free of contamination by human pathogens such as thehepatitis or human immunodeficiency virus. However, use of murinemonoclonal antibodies in some human therapies, have resulted in thedevelopment of an immune response to the “foreign” murine proteins. Thisresponse has been termed a human anti-mouse antibody or HAMA response(Schroff, R. et al. (1985), Cancer Res., 45, 879-885) and is a conditionwhich causes serum sickness in humans and results in rapid clearance ofthe murine antibodies from an individual's circulation. The immuneresponse in humans has been shown to be against both the variable andthe constant regions of murine immunoglobulins.

Recombinant DNA technology can be used to alter antibodies, for example,by substituting specific immunoglobulin regions from one species withimmunoglobulin regions from another species. Neuberger et al. (PatentCooperation Treaty Patent Application No. PCT/GB85/00392) describes aprocess whereby the complementary heavy and light chain variable domainsof an Ig molecule from one species may be combined with thecomplementary heavy and light chain Ig constant domains from anotherspecies. This process may be used to substitute the murine constantregion domains to create a “chimeric” antibody which may be used forhuman therapy. A chimeric antibody produced as described by Neuberger etal. has a human Fc region for efficient stimulation of antibody mediatedeffector functions, such as complement fixation, but still has thepotential to elicit an immune response in humans against the murine(“foreign”) variable regions.

Winter (British Patent Application Number GB2188538A) describes aprocess for altering antibodies by substituting the complementaritydetermining regions (CDRs) with those from another species. This processmay be used to substitute the CDRs from the murine variable regiondomains of a monoclonal antibody with desirable binding properties (forinstance to a human pathogen) into human heavy and light chain Igvariable region domains. These altered Ig variable regions may then becombined with human Ig constant regions to create antibodies which aretotally human in composition except for the substituted murine CDRs. The“reshaped” or “humanized” antibodies described by Winter elicit aconsiderably reduced immune response in humans compared to chimericantibodies because of the considerably less murine components. Further,the half life of the altered antibodies in circulation should approachthat of natural human antibodies. However, as stated by Winter, merelyreplacing the CDRs with complementary CDRs from another antibody whichis specific for an antigen such as a viral or bacterial protein, doesnot always result in an altered antibody which retains the desiredbinding capacity. In practice, some amino acids in the framework of theantibody variable region interact with the amino acid residues that makeup the CDRs so that amino acid substitutions into the human Ig variableregions are likely to be required to restore antigen binding.

Bispecific molecules, (e.g., heteroantibodies) comprising an anti-Fcreceptor portion and an anti-target portion have been formulated andused therapeutically, e.g., for treating cancer (e.g. breast or ovarian)or pathogenic infections (e.g., HIV) (See, e.g., International PatentApplication Publication No. WO 91/05871 entitled BispecificHeteroantibodies With Dual Effector Functions; and International PatentApplication Publication No. WO 91/00360 entitled Bispecific Reagents forAIDS Therapy). In addition, bispecific molecules, which recognizeantigens and antigen presenting cells can be administered to a subjectto stimulate an immune response (See, e.g., International PatentApplication Publication No. WO 92/05793 entitled TargetedImmunostimulation With Bispecific Reagents).

SUMMARY OF THE INVENTION

The present invention provides recombinant and chemically synthesizedmultispecific molecules which target immune cells. The molecules are“multispecific” because they bind to multiple (two or more), distincttargets, one of which is a molecule on the surface of an immune cell,such as an effector cell and/or an antigen presenting cell (APC).

In one embodiment, multispecific molecules of the invention includemolecules comprised of at least one portion which binds to a molecule onan effector cell, such as an Fc receptor, and at least one portion(e.g., two, three, four or more portions) which binds to a differenttarget, such as an antigen on a tumor cell or a pathogen. Therefore,these molecules can be used to induce effector cell-mediated eliminationof a target.

In another embodiment, multispecific molecules of the invention includeantigen “multimer complexes” comprised of multiple (i.e., two or more)portions which bind to a molecule on an antigen presenting cell (APC),such as an Fc receptor, linked to one or more antigens. These multimercomplexes target antigens, such as self-antigens, to APCs to induceand/or enhance internalization (endocytosis), processing and/orpresentation of the antigen by the APC. Therefore, these molecules canbe used to induce or enhance an immune response either in vivo or invitro against a normally non-immunogenic protein, such as aself-antigen.

In another aspect, the invention features multispecific, multivalentmolecules, which minimally comprise an anti-Fc receptor portion, ananti-target portion and optionally an anti-enhancement factor (anti-EF)portion. In preferred embodiments, the anti-Fc receptor portion is anantibody fragment (e.g., Fab or (Fab′)₂ fragment), the anti-targetportion is a ligand or antibody fragment and the anti-EF portion is anantibody directed against a surface protein involved in cytotoxicactivity. In a particularly preferred embodiment, the recombinantanti-FcR antibodies, fragments or ligand are “humanized” (e.g., have atleast a portion of a complementarity determining region (CDR) derivedfrom a non-human antibody (e.g., murine) with the remaining portion(s)being human in origin).

In another aspect, the invention features methods for generatingmultispecific molecules. In one embodiment, both specificities areencoded in the same vector and are expressed and assembled in a hostcell. In another embodiment, each specificity is generated recombinantlyand the resulting proteins or peptides are conjugated to one another viasulfhydryl bonding of the C-terminus hinge regions of the heavy chain.In a particularly preferred embodiment, the hinge region is modified tocontain only one sulfhydryl residue, prior to conjugation.

In yet another aspect, the invention features molecular complexes, whichcomprise two or more binding specificities linked to at least oneantigen. The binding specifities are for a component on the surface ofan antigen presenting cell, which is capable mediating internalizationof the molecular complex when bound by the binding specificities. In apreferred embodiment, the molecular complex comprises three or morebinding specificities, and at least one of the binding specifities arespecific to an Fc Receptor (e.g., FcγRI). In a particularly preferredembodiment, the antigen is non-immunogenic when administered inuncomplexed form.

The invention also features methods for inducing or enhancing an immuneresponse against an antigen in a subject, by administering to thesubject the molecular complex of the invention. Furthermore, theinvention includes methods for immunizing a subject, by administering tothe subject the molecular complex of the invention.

Recombinant antibodies and multispecific molecules generated therefromcan be engineered to have increased affinity and specificity. Further,humanized antibodies are typically less immunogenic when administered toa human. Other features and advantages of the present invention willbecome better understood by reference to the following DetailedDescription and Claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(A-C) shows the nucleotide and amino acid sequences of a portionof the hinge region of a humanized Fcγ RI antibody, H22. [A] that wasaltered to produce a truncated single-sulfhydryl version [B] and thenaltered further to engineer two unique cloning sites [C]. Underlinednucleotides indicate changes from the previous sequence. Overlinednucleotides are the recognition sequences for the indicated restrictionsites.

FIG. 2 is a schematic representation of the heavy chain-EGF fusionexpression construct pJG055.

FIG. 3 is a schematic representation of the generation of anti-Fcreceptor-ligand bispecific molecules.

FIG. 4 is a schematic representation of the flow cytometric assay usedfor testing the activity of the humanized Fcγ receptor- epidermal growthfactor fusion protein.

FIG. 5 is a graph, which plots Mean Fluorescence Intensity (MFI) anindication of the binding of various concentrations of epidermal growthfactor (EGF) fusion protein (H22-EGF fusion) and the fully humanizedbispecific (BsAb) H447 to EGF receptor (EGFR) expressing 1483 cells.

FIG. 6 is a graph, which plots the binding of various concentrations ofthe EGF fusion protein or the BsAb H447 to A431 cells in the presenceand absence of murine antibody M425, which binds EGFR.

FIG. 7 is a graph, which plots the antibody dependent cytotoxicity(ADCC) resulting from the binding of various concentrations of the EGFfusion protein, BsAb H447 or the H425 antibody to A431 cells.

FIG. 8 is a a bar graph which plots the ADCC resulting from the bindingof EGF fusion protein, BsAb H447 or the H425 antibody in the presence ofmedia alone, media containing 25% human serum (HS) or media containing afab fragment of the Fcγ receptor antibody m22.

FIG. 9 is a schematic diagram representing the number of viable A431cells cultured in the presence of various amounts of EGF, H22-EGF, theFab fragment of H22 (H22 Fab), or the F(ab′)₂ fragment of H425 (H425F(ab′)2).

FIG. 10 shows the amino acid sequence of the H22Fd-HRG fusion protein.

FIG. 11 is a histogram indicating the percentage of specific PC-3 orSKBr-3 tumor cell killing resulting from incubation of these cells withinterferon-γ-treated monocytes and a 1:3 or 1:30 dilution of supernatantfrom myeloma cells expressing an H22-heregulin fusion protein.

FIG. 12 is a diagram indicating the percentage of PC-3 tumor cell lysisin the presence of monocytes and in the presence of variousconcentrations of H22-bombesin fusion protein concentrations.

FIG. 13 is a schematic representation of the flow cytometric assay usedfor testing the activity of BsAb 447 generated either by the o-PDM orthe DTNB method.

FIG. 14 is a graph, which plots the MFI of various concentrations ofo-PDM and DTNB derived BsAb 447 to EGFR and FcγRI expressing A431 cells.

FIG. 15 is a graph, which plots the antibody dependent cytotoxicityresulting from the binding of o-PDM and DTNB derived BsAb 447 to A431cells.

FIG. 16(A-B) is a flow chart that depicts the construction oftrispecific antibodies.

FIG. 17 depicts the transformation of a bivalent, bispecific antibodyinto a trivalent, bispecific antibody. The bivalent, bispecificconjugate is reduced and mixed with o-PDM-treated 520C9 Fab′ resultingin the TsAb.

FIG. 18(A-B) depicts a bifunctional fluorescence-activated cell sortingassay for HER2/neu (panel A) and EGFR (panel B).

FIG. 19 is a graph which plots the binding of various concentrations ofantibody, either BsAb or TsAb, to target cells. Mean FluorescenceIntensity (MFI) increases as Ab binding increases. It shows that theTsAb bound both HER2/neu on SKBr-3 cells and soluble FcγRIsimultaneously in a dose-dependent fashion.

FIG. 20 is a graph that shows the TsAb bound both EGFR on A431 cells andsoluble FcγRI simultaneously in a dose-dependent fashion. The assay issimilar to that used in FIG. 19.

FIG. 21 is a graph that shows the TsAb, M22xH425x520C9, and the BsAb,M22x520C9 were capable of inducing ADCC of SKBR-3 cells but the BsAb,M22xH425, was not. Various concentrations of antibodies were incubatedwith SKBR-3 cells and pre-activated PMNs.

FIG. 22 is a graph that shows the TsAb, M22xH425x520C9, and the BsAb,M22xH425 were capable of inducing ADCC of A431 cells but the BsAb,M22x520C9, was not. The assay was performed in a similar manner as theassay in FIG. 21.

FIG. 23(A-B) is a flow chart for a whole blood modulation assay (panelA) and the results from the assay (panel B). This trivalent antibodyrapidly modulates FcγRI from the surface of monocytes.

FIG. 24, panel A, shows the amino acid sequence of oligonucleotidesencoding the wildtype (TT830) and mutant (TT833) tetanus toxin peptides.Panel B is a diagram of an H22Fd-TT fusion protein.

FIG. 25 panels A, B, and C represent flow cytometry analysis resultsshowing binding of MDXH210, Fab22-TT830, and H22-TT833S to FcγRIpositive U937 cells, respectively. The dashed lines represent negativecontrols, the solid lines denote staining with the fusion proteins, andthe dotted lines respresent fusion protein binding blocked by murine mAb22 F(ab′)₂.

FIG. 26 is a schematic diagram showing the mean fluorescence intensityresulting from incubation of various amounts of the fusion proteinsMDXH210, FAb22-TT830, and Fab22-TT833S to FcγRI positive U937 cells.

FIG. 27 is a graphic representation of the proliferation of T cellsincubated with irradiated monocytes and various concentrations of TT830,Fab22-TT830, TT, or TT947, showing that the fusion protein Fab22-TT830enhances the presentation of the Th epitope by about 1000 fold ascompared to TT830.

FIG. 28 represents a histogram showing the proliferation of T cellsincubated with TT830 at 1000 nM or FAb22-TT830 at 10 nM and monocytes,preincubated or not with saturating amounts of mAb 22 F(ab′)2 prior toaddition of the T cells and the antigen.

FIG. 29 represents a histogram showing the proliferation of T cellsincubated with monocytes and Fab22-TT830 at 5 nM or TT830 at 1000 nM inthe absence (control) or presence of IgG.

FIG. 30(A-B), panels A and B, are graphic representations showing theconcentration of IFN-γ (panel A) and IL-4 (panel B) in the supernatantof T cells cultured for 2 days with monocytes and various concentrationsof TT830 or Fab22-TT830.

FIG. 31 is a graphic representation depicting the proliferation of Tcells incubated with monocytes and various concentrations of TT833S,Fab22-TT833S, or TT830.

FIG. 32 is a graphic representation of the proliferation of T cellsincubated for 2 days with TT830 and monocytes, preincubed overnight withvarious concentrations of TT833S.

FIG. 33 is a graphic representation of the percent inhibition ofproliferation of T cells incubated for 2 days with TT830 and monocytes,preincubated overnight with various concentrations of TT833S orFAb22-TT833S.

FIG. 34 is a histogram representing the proliferation of T cellsincubated for 2 days with monocytes, which were first incubated withTT830 for 4 hours (Pre-pulse) and then incubated overnight with 10 μMTT833S or 0.1 μM Fab22-TT833S (Chase) prior to addition of the T cells.

FIG. 35 is a histogram representing the concentration of interferon-γ(IFN-γ) and IL-4 in the supernatant of T cells cultured with monocytesand TT830, FAb22-TT830, TT833S, and Fab22-TT833S.

FIG. 36 is a graphic representation of the proliferation of T cellsstimulated for one day with monocytes in medium alone, with TT833S, orwith Fab22-TT833S and then restimulated with monocytes and variousconcentrations of TT830 for two days, indicating that TT833S andFab22-TT833S do not lead to T cell anergy.

FIG. 37 is a graphic representation of two expression constructsencoding single chain bispecific molecules having one bindingspecificity for an FcγRI (H22) and one binding specificity for acarcinoembryonic antigen (CEA) (constructs 321 adn 323) and oneexpression construct encoding a single chain antibody having one bindingspecificity for an FcγRI. The coding regions are under the control ofthe CMV promoter (CMV Pr). In addition to the variable regions from theheavy (VH) and light chains (VL) of the antibodies, the proteins encodedby these constructs are fused to a peptide from c-myc (c-myc) and to ahexa-histidine peptide (H-6).

FIG. 38 shows a histogram indicating the level of binding of the singlechain bispecific molecules H22-anti-CEA encoded by the expressionconstructs 321 (321-A5 and 321-B4) and 323 (323-B2 and 323-C4) and thesingle chain H22 antibody encoded by the construct 225 (225-C2) asmeasured by bispecific ELISA.

FIG. 39(A-C) shows the nucleic acid sequence of the single chainhumanized anti-FcγRI antibody and the amino acid sequence encoded by thenucleic acid.

FIG. 40(A-D) shows the nucleic acid sequence of the single chainbispecific molecule having one binding specificity for the FcγRI and onebinding specificity for CEA and the amino acid sequence encoded by thenucleic acid.

FIG. 41 is a schematic representation of the formation of multimercomplexes made up of multiple Fab′ fragments linked to an antigen.

FIG. 42(A-B) is a non-reducing SDS-PAGE gel showing a purified M22(ab′)2 multimer (lane 1) and a chemically linked M22 Fab′ multimercomplex (lane 2). FIG. 42(B) is a graph showing that incubation with M22F(ab′)3+ results in up to 50% reduction in CD64 expression on thesurfaces of macrophages in a dose dependent fashion.

FIG. 43(A) shows that high titers of M22-specific antibody weregenerated in all of the CD64 transgenic mice immunized three times,compared to their nontransgenic littermates. FIG. 43(B) shows thatimmunizing FcγRI (CD64) transgenic mice with as little as 0.25 mg of amultimer complex still leads to a detectable immune response.

FIGS. 44(A) and 44(B) are graphs showing the immune response of FcγRICD64) transgenic and nontransgenic mice as judged by anti-520C9 andanti-M22 titers. The mice were immunized with a Fab′fragment of a murineantibody, 520C9, coupled to a M22 multimer complex.

FIG. 45(A) shows a map of an expression vector encoding a geneticallylinked multimer complex containing two H22 sFv regions linked to oneM32.2 sFv region linked to an antigen (H22(2x)-32.2-antigen complex).FIG. 45(B) is a drawing of the resulting multimer complex.

FIG. 46 is a bar graph showing the ability of M22 Fab x M22 Fab x M32and H22sFv-H22sFv-32.2sFv-gp75 multimer complexes to bind to FcγRIefficiently and induce internalization.

FIG. 47(A) shows a map of an expression vector encoding a geneticallylinked multimer complex containing two H22 sFv regions linked togetherto an antigen (H22(2x)-antigen multimer complex). FIG. 47(B) is adrawing of the resulting multimer complex.

FIG. 48 is a graph showing the results of a binding competition assayusing H22 Fab′, H22sFv2-EGF multimer and H22 Fab2 multimer. Results areshown in terms of binding inhibition of an M22-phycoerythrin conjugateby these multimers.

FIG. 49(A) shows a map of an expression vector encoding a geneticallylinked multimer complex containing three H22 sFv fragments linked to anantigen (H22(3x)-antigen multimer complex). FIG. 49(B) is a drawing ofthe resulting protein.

FIG. 50 is a bar graph showing the ability of an H22(3x)-CEA multimercomplex to induce internalization of FcγRI compared to other multimercomplexes.

DETAILED DESCRIPTION

Multispecific Molecules

The instant invention relates to recombinant and chemically synthesizedmultispecific molecules which target immune cells. The molecules are“multispecific” because they bind to multiple (two or more), distincttargets, one of which is a molecule on the surface of an immune cell. Inone embodiment, multispecific molecules of the invention includemolecules comprised of at least one portion which binds to a molecule onan effector cell, such as an Fc receptor, and at least one portion(e.g., two, three, four or more portions) which binds to a differenttarget, such as an antigen on a tumor cell or a pathogen. In anotherembodiment, multispecific molecules of the invention include antigen“multimer complexes” comprised of multiple (i.e., two or more) portionswhich bind to a molecule on an antigen presenting cell (APC), such as anFc receptor, linked to one or more antigens. These multimer complexestarget antigens, such as self-antigens, to APCs to induce and/or enhanceinternalization (endocytosis), processing and/or presentation of theantigen by the APC. Therefore, these molecules can be used to induce orenhance an immune response either in vivo or in vitro against a normallynon-immunogenic protein, such as a self-antigen.

As used herein to describe the multispecific molecules of the presentinvention, the term a “portion which binds to” is used interchangeablywith the term a “binding specificity for.” Both of these terms refer toregions of the multispecific molecules which bind to a target epitope.As described in detail below, these portions or binding specificitiesinclude any compound capable of binding to a target epitope including,but not limited to antibodies, antibody fragments (e.g., an Fab, Fab′,F(ab′)₂, Fv, or a single chain Fv) and mimetics thereof (e.g., peptide,chemical and organic mimetics which “mimic” the binding of an antibodyor antibody fragment). Suitable antibodies and antibody fragmentsinclude, but are not limited to murine, humanized (chimeric), singlechain, and human monoclonal antibodies and fragments thereof. In aparticular embodiment, the binding specificities include one or moreantibodies selected from H22 (ATCC Deposit No. CRL 11177), M22 (ATCCDeposit No. HB 12147), M32.2 (ATCC Deposit No HB 9469), and antigenbinding fragments thereof, each of which binds to FcγRI. Humanizedantibody H22, and its murine equivalent, M22, provide the advantage ofbinding to FcγRI outside the natural ligand (IgG) binding site and,thus, are not blocked or competed off by endogenous ligand, whenadministered in vivo.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., an Fc receptor). It has been shown that the antigen-bindingfunction of an antibody can be performed by fragments of a full-lengthantibody. Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR).Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.These antibody fragments are obtained using conventional techniquesknown to those with skill in the art, and the fragments are screened forutility in the same manner as are intact antibodies.

Antigen Multimer Complexes

Antigen multimer complexes of the invention include multiple portions orbinding specificities which target a component on the surface of anantigen presenting cell (APC cell surface component), linked to one ormore antigens. The multiple binding specificities can bind to the sameor different epitopes on the APC cell surface component, or to differentAPC cell surface components. In a preferred embodiment, the multimercomplex includes at least three binding specificities for APC cellsurface component(s). Suitable components on APCs for targeting includethose which, when bound by the binding specificity, mediate itsinternalization, so that the antigen linked to the binding specificityis efficiently internalized and presented by the APC.

As used herein, the term “antigen presenting cell” or “APC” refers to animmune cell capable of internalizing and processing an antigen, so thatantigenic determinants are presented on the surface of the cell asMHC-complexes, in a manner capable of being recognized by the immunesystem (e.g., class II-MHC restricted helper T lymphocytes). The tworequisite properties that allow a cell to function as an APC are theability to process endocytosed antigens and the expression of class IIMHC gene products. The best defined APCs for helper T cells includemononuclear phagocytes (e.g., macrophages), B lymphocytes, dendriticcells, Langerhans cells of the skin and, in humans, endothelial cells.

In a preferred embodiment, the APC cell surface component targeted bythe binding specificity is an Fc receptor, typically FcγR. Therefore, ina particular embodiment of the invention, the multimer complex includesmultiple binding specificities (e.g., two or more, preferably three ormore) which target different epitopes on FcγRI. Alternatively, themultimer can include multiple binding specificities which target thesame epitope on FcγRI. However, other suitable APC cell surfacecomponents also can be targeted. Such components can be identified, forexample, by immunizing an animal (e.g., mice transgenic for human FcγRI)with APCs and determining cell surface components bound by sera takenfrom the animal, e.g., using standard (e.g., Western blot) assays. TheseAPC cell surface components can then be tested for their ability tointernalize a compound which binds to the component.

Accordingly, in one embodiment, the multimer complex includes containsat least one antibody or fragment thereof (e.g., an Fab, Fab′, F(ab′)₂,Fv, or a single chain Fv) which binds to an Fc receptor, such as a humanIgG receptor, e.g., an Fc-gamma receptor (FcγR), such as FcγRI (CD64),FcγRII(CD32), and FcγRIII (CD16). A preferred Fcγ receptor is the highaffinity Fcγ receptor, FcγRI. However, other Fc receptors, such as humanIgA receptors (e.g FcαRI) also can be targeted. The Fc receptor islocated on the surface of an APC, e.g., a monocyte or macrophage. In aparticular embodiment, the multimer complex binds to an Fc receptor at asite which is distinct from the immunoglobulin (e.g., IgG or IgA)binding site of the receptor. Therefore, the binding of the multimercomplex is not blocked by physiological levels of immunoglobulins.Preferred humanized anti-FcγR monoclonal antibodies are described in PCTapplication WO 94/10332 and U.S. Pat. No. 4,954,617, the teachings ofwhich are fully incorporated herein by reference).

As described in the Examples herein, particular humanized and murinemonoclonal anti- FcγRI antibodies and antibody fragments suitable foruse in the antigen multimer complexes of the invention include, but arenot limitd to, humanized antibody H22 (ATCC Deposit No.CRL11177), itsmurine counterpart, M22 (ATCC Deposit No. HB12147), and murine M32.2(ATCC Deposit No. HB 9469), as well as antigen binding fragments ofthese antibodies.

Multimer complexes of the invention which target APCs further includeone or more antigens. The term “antigen”, as used herein, refers to anymolecule (e.g., protein, peptide, carbohydrate etc.) which is capable ofbeing recognized by an immune cell (i.e., eliciting an immune response,such as a T cell-mediated immune response). Antigens include “selfantigens” or “autoantigens” which are normally present in a host, andwhich normally do not elicit an immune response from the host (becausethe immune system recognizes them as “self” and not “foreign”). Incertain disorders, antigens which are not normally present in a hostand, therefore, should elicit an immune response by a host's immunesystem, do not. In other words, they “escape” recognition by a host'simmune system. Such antigens include, for example, certain tumorantigens and pathogenic (e.g., viral and bacterial) antigens. In thesesituations, it may be desirable to modify the antigen in a way thatinduces or enhances its recognition by immune cells, so that the antigenor cell/organism which produces the antigen is eliminated from the host.In other words, the antigen is modified so that it is no longerrecognized by immune cells as a “self antigen.”

Antigen multimer complexes of the invention can be prepared bychemcially linking one or more antigens to multiple (two or more)binding specificities for a component on an APC, as described herein,using standard cross-linking reagents and conjugation protocols wellknown in the art. Alternatively, the antigen multimer complexes can berecombinantly produced as a single fusion protein, also as describedherein.

Accordingly, in yet another embodiment, the present invention provides anucleic acid encoding an antigen multimer complex. Typically, thenucleic acid is operatively linked, within an expression vector, to apromoter and other genetic regulatory sequences which control expressionof the multimer complex. A nucleic acid is “operably linked” when it isplaced into a functional relationship with another nucleic acidsequence. For instance, a promoter or enhancer is operably linked to acoding sequence if it affects the transcription of the sequence. Withrespect to transcription regulatory sequences, operably linked meansthat the DNA sequences being linked are contiguous and, where necessaryto join two protein coding regions, contiguous and in reading frame. Forswitch sequences, operably linked indicates that the sequences arecapable of effecting switch recombination.

The term “vector”, as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinantexpression vector has been introduced. It should be understood that suchterms are intended to refer not only to the particular subject cell butto the progeny of such a cell. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

Antigen multimer complexes of the invention can be used to induce orenhance internalization, processing and presentation of an antigen by animmune cell (e.g., an APC). Accordingly, the present invention furtherprovides a method of inducing or enhancing an immune response against anantigen in a subject by administering to the subject an effective amountof an antigen multimer complex. For example, an antigen multimer complexcan be administered to induce an immune response against a self-antigen(including tumor antigens not recognized by an immune system), or toenhance an immune response against an antigen, such as a tumor antigenor a component of a pathogen. As such, the antigen multimer complexesalso can be used as vaccines to immunize a host.

Multispecific Molecules which Target Effector cells

Multispecific molecules of the invention also include molecules designedto target effector cells, for example, to cause effector cell-mediatedelimination of a target cell, pathogen, allergen or other entity.Accordingly, the invention provides, in another aspect, a multispecificmolecule comprised of at least one portion which binds to a component onan effector cell, typically an Fc receptor, and at least one portion(e.g., two, three, four or more portions) which binds to a differenttarget, such as an antigen on a tumor cell or a pathogen.

An “effector cell”, as used herein refers to an immune cell. Specificeffector cells express specific Fc receptors and carry out specificimmune functions. For example, monocytes, macrophages, neutrophils anddendritic cells, which express FcγRI are involved in both specifickilling of target cells and presenting antigens to other components ofthe immune system. The expression of a particular FcR on an effectorcell can be regulated by humoral factors such as cytokines. For example,expression of FcγRI has been found to be up-regulated by interferongamma (IFN-γ). This enhanced expression increases the cytotoxic activityof FcγRI cells against targets.

Multispecific molecules which target effector cells have one or morebinding specificities or portions which bind to a component on aneffector cell, such as an Fc receptor. In one embodiment, the bindingspecificity is provided by an antibody or antibody fragment, aspreviously described herein. In a particular embodiment, the antibody orantibody fragment is human or is “humanized” (i.e. derived from a humanantibody, but having at least a portion of a complementarity determiningregion (CDR) derived from a non-human antibody, the portion beingselected to provide specificity of the humanized antibody for e.g., ahuman Fc receptor). The humanized antibody has CDRs derived from anon-human antibody and the remaining portions of the antibody moleculeare human.

The antibody may be whole, i.e. having heavy and light chains or anyfragment thereof, e.g., Fab or (Fab′)₂ fragment. The antibody furthermay be a light chain or heavy chain dimer, or any minimal fragmentthereof such as a Fv or a single chain construct as described in Ladneret al. (U.S. Pat. No. 4,946,778, issued Aug. 7, 1990), the contents ofwhich is expressly incorporated by reference. The humanized antibody orfragment may be any human antibody capable of retaining non-human CDRs.The preferred human antibody is derived from known proteins NEWM and KOLfor heavy chain variable regions (VHs) and REI for Ig kappa chain,variable regions (VKs).

The portion of the non-human CDR inserted into the human antibody isselected to be sufficient for allowing binding of the humanized antibodyto the Fc receptor. A sufficient portion may be selected by inserting aportion of the CDR into the human antibody and testing the bindingcapacity of the created humanized antibody using the enzyme linkedimmunosorbent assay (ELISA).

All of the CDRs of a particular human antibody may be replaced with atleast a portion of a non-human CDR or only some of the CDRs may bereplaced with non-human CDRs. It is only necessary to replace the numberof CDRs required for binding of the humanized antibody to the Fereceptor. A non-human CDR derived from a murine monoclonal antibody(mab), mab 22, is described in International Patent ApplicationPublication No. WO 94/10332, the contents of which are fullyincorporated herein by reference. The mab 22 antibody is specific to theFc receptor and further is described in U.S. Pat. No. 4,954,617, issuedSep. 4, 1988, the contents of which are also expressly incorporated byreference. The humanized mab 22 antibody producing cell line wasdeposited at the American Type Culture Collection on Nov. 4, 1992 underthe designation HA022CL1 and has the accession no. CRL 11177.

An antibody can be humanized by any method, which is capable ofreplacing at least a portion of a CDR of a human antibody with a CDRderived from a non-human antibody. Winter describes a method which maybe used to prepare the humanized antibodies of the present invention (UKPatent Application GB 2188638A, filed on Mar. 26, 1987), the contents ofwhich is expressly incorporated by reference. The human CDRs may bereplaced with non-human CDRs using oligonucleotide site-directedmutagenesis as described in International Patent Application PublicationNumber: WO 94/10332 entitled, Humanized Antibodies to Fc Receptors forImmunoglobulin G on Human Mononuclear Phagocytes.

In addition to an anti-Fc receptor portion, the multispecific moleculescan comprise an “anti-target portion”, i.e. an antibody, a functionalantibody fragment or a ligand that recognizes and binds a pathogen(e.g., virus, bacteria, fungi), a pathogen infected cell, a cancer ortumor cell (e.g., breast, ovarian, prostate, etc.) or other unwantedcell in a subject (e.g., a human or animal) or an antigen or modifiedform thereof. Additionally, the target portion may comprise or bedirected against an antigen. A preferred embodiment contains an antigenthat can be used to stimulate the immune system, for example, ininstances of chronic infection, to deplete antigen in the circulation,and to treat tumors. A particularly preferred embodiment has an antigenthat is attached to a multivalent molecule containing an anti-FcRantibody.

In a specific embodiment of the invention, the multispecific moleculecontains a ligand. The ligand can be any ligand that interacts with amolecule. In a preferred embodiment, the ligand binds a protein, e.g., asurface protein on a target cell, such as a cancer cell. Preferredligands include ligands to receptors, such as growth or differentiationfactors. For example, a multivalent molecule can comprise an epidermalgrowth factor, or at least a portion or modified form that is capable ofinteracting with a receptor, e.g., an epidermal growth factor receptor.In another preferred embodiment of the invention, the ligand is a smallpeptide, such as bombesin, gastrin-releasing peptide (GRP), litorin,neuromedin B, or neuromedin C. The sequences of the peptides can befound, e.g., in U.S. Pat. No. 5,217,955, the content of which isincorporated herein by reference. The ligand can also be a modified formof any of these peptides. The modification can increase binding to thereceptor, decrease binding, or not affect the binding to a receptor. Themodification of the ligand can also transform an agonist into anantagonist, such that the ligand inhibit rather than stimulate cellproliferation. The modification of the ligand can be an addition, adeletion, a substitution, or a modification of at least one amino acid.

In a specific embodiment of the invention, a multivalent or bispecificmolecule comprises an antigen. As used herein, the term “antigen” meansany natural or synthetic immunogenic substance, a fragment or portion ofan immunogenic substance, a peptidic epitope, or a hapten. The term“antigen” also includes substances which are non-immunogenic inuncomplexed form, but are immunogenic when complexed. The term“uncomplexed” includes substances which are not linked to form amolecular complex of the present invention. The term “complexed”includes substances which are linked to form a molecular complex of thepresent invention.

In one embodiment of the invention, a bi- or multispecific molecule isemployed to target an antigen to the cell to enhance the processes ofinternalization and presentation by these cells, and utlimately, tostimulate an immune response therein. In a specific embodiment, thebispecific binding agent specifically binds the antigen (eitherdirectly, to an epitope of the antigen, or indirectly, to an epitopeattached to the antigen) and, at the same time, binds a surface receptorof an antigen-presenting cell which can internalize antigen forprocessing and presentation. In another embodiment, the antigen islinked to the multi- or bispecific molecule and at the same time binds asurface receptor of an antigen-presenting cell. The receptor-bindingcomponent of these bi- or multispecific molecule (and thus the bi- ormultispecific molecule, itself) binds the receptor of theantigen-presenting cell. In some instances, binding of the moleculeoccurs without the molecule substantially being blocked by the naturalligand for the receptor. As a result, targeting of the antigen to thereceptor will not be prevented by physiological levels of the ligand andthe targeted receptor will remain capable of binding the ligand andfunctioning.

One type of antigen can be an allergen. An “allergen” refers to asubstance that can induce an allergic or asthmatic response in asusceptible subject. The list of allergens is enormous and can includepollens, insect venoms, animal dander dust, fungal spores and drugs(e.g. penicillin). Examples of natural, animal and plant allergensinclude proteins specific to the following genuses: Canine (Canisfamiliaris); Dermatophagoides (e.g. Dermatophagoides farinae); Felis(Felis domesticus); Ambrosia (Ambrosia artemiisfolia; Lolium (e.g.Lolium perenne or Lolium multiflorum); Cryptomeria (Cryptomeriajaponica); Alternaria (Alternaria alternata); Alder; Alnus (Alnusgultinosa); Betula (Betula verrucosa); Quercus (Quercus alba); Olea(Olea europa); Artemisia (Artemisia vulgaris); Plantago (e.g. Plantagolanceolata); Parietaria (e.g. Parietaria officinalis or Parietariajudaica); Blattella (e.g. Blattella germanica); Apis (e.g. Apismultiflorum); Cupressus (e.g. Cupressus sempervirens, Cupressusarizonica and Cupressus macrocarpa); Juniperus (e.g. Juniperussabinoides, Juniperus virginiana, Juniperus communis and Juniperusashei) Thuya (e.g. Thuya orientalis); Chamaecyparis (e.g. Chamaecyparisobtusa); Periplaneta (e.g. Periplaneta americana); Agropyron (e.g.Agropyron repens); Secale (e.g. Secale cereale); Triticum (e.g. Triticumaestivum); Dactylis (e.g. Dactylis glomerata); Festuca (e.g. Festucaelatior); Poa (e.g. Poa pratensis or Poa compressa); Avena (e.g. Avenasativa); Holcus (e.g. Holcus lanatus); Anthoxanthum (e.g. Anthoxanthumodoratum); Arrhenatherum (e.g. Arrhenatherum elatius); Agrostis (e.g.Agrostis alba); Phleum (e.g. Phleum pratense); Phalaris (e.g. Phalarisarundinacea); Paspalum (e.g. Paspalum notatum); Sorghum (e.g. Sorghumhalepensis); and Bromus (e.g. Bromus inermis).

Many allergens are found in airborne pollens of ragweed, grasses, ortrees, or in fungi, animals, house dust, or foods. As a class, they arerelatively resistant to proteolytic digestion. Preferable allergens arethose which bind to IgE on mast cells and basophils, thereby causing atype I anaphylaxis hypersensitivity reaction. When at least onespecificity of the multivalent agent is for an epitope of the highaffinity Fc receptor that is outside the ligand binding domain for IgG,this bispecific binding agent can decrease hypersensitivity in asubject. This is accomplished when the bispecific binding agent competesfor an IgE-binding allergen before the allergen binds to IgE on a mastcell or basophil, thereby reducing the possibility of a type Ihypersensitivity reaction. In addition, as a result of directingallergen to FcγR, a state of T cell tolerance to the allergen may beinduced which interferes with IgE-mediated type I reactions. Tolerancecan be accomplished by inducing IgG which competes with IgE for bindingto allergen using doses of allergen substantially lower than thosecurrently used.

In some cases, it may be desirable to couple a substance which is weaklyantigenic or nonantigenic in its own right (such as a hapten) to acarrier molecule, such as a large immunogenic protein (e.g., a bacterialtoxin) for administration. In these instances, the bispecific bindingreagent can be made to bind an epitope of the carrier to which thesubstance is coupled, rather than an epitope of the substance itself.

The antigen that can be linked either directly, or indirectly, to amulti- or bispecific molecule of the invention can be soluble orparticulate; it may carry B cell epitopes, T cell epitopes or both. Theantigen can be bacterial, viral or parasitic in origin. Often, theantigen will comprise a component of the surface structure of apathogenic organism. For example, the antigen can comprise a viralsurface structure such as an envelope glycoprotein of humanimmunodeficiency virus (HIV) or the surface antigen of hepatitis virus.In addition, the antigen can be associated with a diseased cell, such asa tumor cell, against which an immune response may be raised fortreatment of the disease. The antigen can comprise a tumor-specific ortumor-associated antigen, such as the Her-2/new proto-oncogene productwhich is expressed on human breast and ovarian cancer cells (Slamon etal. (1989) Science 244:707).

The cells of a subject can be exposed in vitro or in vivo to themultivalent molecules of the invention. The multivalent molecule can beused to target an antigen to antigen-presenting cells in culture.Immunocompetent cells are separated and purified from patient blood. Thecells are then exposed to a multivalent molecule comprising the antigenor the cells can be exposed to the antigen together with a multivalentmolecule having a binding specificity for the antigen. Targetedantigen-presenting cells will process the antigen and present fragmentson their surface. After stimulation, the cells can be returned to thepatient.

The method of this invention can be used to enhance or reinforce theimmune response to an antigen. For example, the method is valuable forthe treatment of chronic infections, such as hepatitis and AIDS, wherethe unaided immune system is unable to overcome the infection. It canalso be used in the treatment of the acute stages of infection whenreinforcement of immune response against the invading organism may benecessary.

The method can be used to reduce the dose of antigen required to obtaina protective or therapeutic immune response or in instances when thehost does not respond or responds minimally to the antigen. Althoughgenerally desirable, the lowering of effective dose can be especiallydesirable when the antigen is toxic to the host such as is the case forallergies. Methods and uses for using bi- or multispecific moleculescomprising an antigen or comprising an ligand, e.g., an antibodyinteracting with an antigen, are further described in the published PCTapplication PCT/US91/07283.

In another embodiment of the invention, a multispecific moleculecomprises an antigen that has been modified, such that its effect on Tcell activation is modified upon presentation of the modified antigen tothe T cell by an antigen presenting cell. Allan et al. have in factshown that substitution of one or more amino acids of a peptide thatstimulates T cells, e.g., stimulates T cell proliferation, can result inan antigen which fails to stimulate the T cell or which induces anergyin the T cell. Such modified peptides are termed Altered Peptide Ligands(APL). Accordingly, such APLs can be linked to bispecific ormultispecific molecules having at least one binding specificity for theFcγRI. Upon phagocytosis of these molecules by antigen presenting cellsand presentation to T cells, the proliferation of the T cells may beinhibited or anergized. Accordingly, administration to a subject of amultispecific molecule comprising (a) at least one altered peptide of anantigen which normally stimulates T cells, but which upon modificationinduces anergy of the T cells, and (b) at least one anti-FcγRI antibodywill result in induction of tolerance of the subject to the antigen.Thus, such multi- or bispecific molecules can be used to tolerize asubject to a variety of antigens, e.g., auto-antigens. Thus, dependingon the antigen used, the methods of the invention provide methods forincreasing an immune response, i.e., by using an antigen whichstimulates T cells, and the invention also provides methods for reducingan immune response, either by inhibiting T cell stimulation or byinducing anergy of the T cells.

The multispecific, multivalent molecules of the invention may alsoinclude an “anti-enhancement factor (anti-EF) portion”. The“anti-enhancement factor portion” can be an antibody, functionalantibody fragment or a ligand that binds to an antigen and therebyresults in an enhancement of the effect of the anti-Fc receptor portionor the anti-target portion. The “anti-enhancement factor portion” canbind an Fc receptor or a target. A multivalent molecule comprising ananti-target portion that binds to one target cell antigen and ananti-enhancement factor portion that binds to a different target antigenis particularly useful where the target cell undergoes antigenmodulation or antigenic variation (e.g., as has been described forcertain parasites (such as trypanosomes). Alternatively, theanti-enhancement factor portion can bind an entity that is differentfrom the entity to which the anti-target or anti-Fc receptor portionbinds. For example, the anti-enhancement factor portion can bind acytotoxic T-cell (e.g. via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 orother immune cell that results in an increased immune response againstthe target).

Methods for Making Multispecific Molecules

The multispecific molecules described above can be made by a number ofmethods. For example, both specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the multi-specific molecule is a ligand x fabfusion protein as described in the following Example 2. A bispecificmolecule of the invention can also be a single chain bispecificmolecule, such as a single chain bispecific antibody, a single chainbispecific molecule comprising one single chain antibody and a ligand,or a single chain bispecific molecule comprising two ligands.Multivalent molecules can also be single chain molecules or may compriseat least two single chain molecules. Methods for preparing bi- ormultivalent antibodies are for example described in U.S. Pat. No.5,260,203; U.S. Pat. No. 5,455,030; U.S. Pat. No. 4,881,175; U.S. Pat.No. 5,132,405; U.S. Pat. No. 5,091,513; U.S. Pat. No. 5,476,786; U.S.Pat. No. 5,013,653; U.S. Pat. No. 5,258,498; and U.S. Pat. No.5,482,858.

Binding of the single chain molecules to their specific targets can beconfirmed by bispecific ELISA as described in the Examples herein.

Alternatively, each specificity of a multispecific molecule can begenerated separately and the resulting proteins or peptides conjugatedto one another. For example, two humanized antibodies can be conjugatedvia sulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particularly preferred embodiment, the hinge region ismodified to contain an odd number of sulfhydryl residues, preferablyone, prior to conjugation.

The bispecific molecules of the present invention can be prepared byconjugating the anti-FcR and anti-target portions using methodsdescribed in the following Example or those well-known in the art. Forexample, a variety of coupling or cross-linking agents can be used forcovalent conjugation. Examples of cross-linking agents include proteinA, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA),N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinirmidyl 4-(N-maleimidomethyl) cyclohaxane-1-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686;Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methodsinclude those described by Paulus (Behring Ins. Mitt. (1985) No. 78,118-132); Brennan et al. (Science (1985) 229:81-83), and Glennie et al.(J. Immunol. (1987) 139: 2367-2375). Preferred conjugating agents areSATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford,Ill.).

Therapeutic Uses for Multispecific Molecules

Based on their ability to bind FcR bearing immune cells and specifictarget cells, a specific multispecific molecule can be administered to asubject to treat or prevent a variety of diseases or conditions,including: cancer (e.g., breast, ovarian, small cell carcinoma of thelung), pathogenic infections (e.g., viral (such as HIV)), protozoan(such as Toxoplasma gondii), fungal (such as candidiasis); anautoimmunity (e.g. immune thrombocytopenia purpura and systemic lupus).The multispecific multivalent can also be administered prophylacticallyto vaccinate a subject against infection by a target cell.

For use in therapy, an effective amount of an appropriate multispecificmolecule can be administered to a subject by any mode that allows themolecules to exert their intended therapeutic effect. Preferred routesof administration include oral and transdermal (e.g., via a patch).Examples of other routes of administration include injection(subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal,etc.). The injection can be in a bolus or a continuous infusion.

A multispecific molecule can be administered in conjunction with apharmaceutically acceptable carrier. As used herein, the phrase“pharmaceutically acceptable carrier” is intended to include substancesthat can be coadministered with a multispecific molecule and allows themolecule to perform its intended function. Examples of such carriersinclude solutions, solvents, dispersion media, delay agents, emulsionsand the like. The use of such media for pharmaceutically activesubstances are well known in the art. Any other conventional carriersuitable for use with the molecules falls within the scope of theinstant invention.

The language “effective amount” of a multispecific molecules refers tothat amount necessary or sufficient to realize a desired biologiceffect. For example, an effective amount of a multispecific molecule, inwhich the anti-target portion recognizes a pathogenic cell could be thatamount necessary to eliminate a tumor, cancer, or bacterial, viral orfungal infection. The effective amount for any particular applicationcan vary depending on such factors as the disease or condition beingtreated, the particular multispecific molecule being administered, thesize of the subject, or the severity of the disease or condition. One ofordinary skill in the art can empirically determine the effective amountof a particular multispecific molecule without necessitating undueexperimentation.

The following invention is further illustrated by the followingexamples, which should not be construed as further limiting. Thecontents of all references, pending patent applications and publishedpatents, cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLES Example 1

Production of Bispecific Antibody Comprising Murine or HumanizedAntibodies Specific for an Fc Receptor and an Anti-her 2 neu Antibody

Monoclonal Antibodies

The anti-FcγRI monoclonal antibodies (mAbs), M22, M32.2 and 197 werepurified from hybridoma supernatant by ion exchange chromatography andDZ33, a human anti-HIV-1 IgG1 mAb, was purified from hybridomasupernatant by protein A affinity chromatography (Pharmacia, Piscataway,N.J.) and gel filtration. M32.2 was deposited at the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, on Jul.1, 1987 and has been designated with ATCC Accession No. HB9469.

Cell Lines

The murine myeloma NSO (ECACC 85110503) is a non-Ig synthesizing lineand was used for the expression of recombinant mAbs. NSO cells werecultivated in DMEM plus 10% fetal bovine serum (FBS, Gibco, Paisley,U.K.). SKBR-3 is a human breast carcinoma cell line which overexpressesthe HER2/neu protooncogene (ATCC, Rockville, Md.) and was cultivated inIscove's Modified Dulbecco's Medium (IMDM, Gibco, Grand Island, N.Y.).U937 is a monocytoid cell line that expresses FcγRI and was obtainedfrom ATCC and grown in RPM-1640 plus 10% FBS (Gibco, Grand Island,N.Y.).

Cloning Murine Immunoglobulin V Region Genes

Cytoplasmic RNA from the murine hybridoma 22 was prepared as describedin Favaloro et al. (Favaloro, J., R. Treisman and R. Kamen (1982)Transcription maps of polyoma-specific RNA: analysis by two-dimensionalS 1 gel mapping. Meth. Enzymol. 65:718). The Ig V region cDNAs were madefrom RNA via reverse transcription initiated from primers CG1FOR andCK2FOR as described in International Patent Application PublicationNumber WO 94/10332 entitled, Humanized Antibodies to Fc Receptors forImmunoglobulin G on Human Mononuclear Phagocytes. The cDNA synthesis wasperformed under standard conditions using 100 U MMLV reversetranscriptase (Life Technologies, Paisley, UK). The V_(H) and V_(κ)cDNAs were amplified by PCR, (Orlandi, R., D. H. Güssow, P. T. Jones andG. Winter (1989) (Cloning immunoglobulin variable domains for expressionby the polymerase chain reaction), Proc. Natl. Acad. Sci. USA 86:3833),using the cDNA primers in concert with SH2BACK and VK7BACK as describedin International Patent Application Publication Number WO 94/10332.Amplified V_(H) and V_(κ) DNA were purified, cloned into M13, andsequenced by the dideoxy method using. T7 DNA polymerase (Pharmacia,Piscataway, N.J.).

Construction of Chimeric Antibody Genes

To facilitate cloning of murine V region DNA into expression vectors,restriction sites were placed in close proximity to the termini of bothM22 V region genes. For V_(H), a 5′ PstI site and a 3′ BstEII site wereintroduced into a cloned murine V_(H) gene by PCR using VH1BACK andVH1FOR (Id.). For V_(κ) a 5′ PvuII site and a 3′ Bgl II site wereintroduced into a cloned murine V_(κ) gene by PCR using primers VK1BACKand VK1FOR (Id.). In some instances, these primers changed one or moreamino acids from those naturally occurring. These V region genes (ChVHand ChVK) were cut with the appropriate restriction enzymes and clonedinto M13VHPCR1 and M13VKPCR1 (Id.) which contain an Ig promoter, signalsequence and splice sites. The DNA were excised from M13 asHindIII-BamHI fragments and cloned into the expression vectors pSVgptand pSVhyg containing human IgG1, (Takahashi, N. et al., (1982),Structure of human immunoglobulin gamma genes: implications forevolution of a gene family, Cell, 29:671), and human kappa constant,(Hieter, R.A. et al., (1980) Cloned human and mouse kappa immunoglobulinconstant and J region genes conserve homology in functional segments,Cell 22:197), region genomic DNA.

Construction of Humanized Antibody Genes

Two humanized heavy chains were constructed and were based on humanV_(H)s of NEWM, (Poljak, R.J. et al., Amino acid sequence of the V_(H)region of a human mycloma immunoglobulin, (IgG New), Biochemistry,16:3412), and KOL,(Marquat, M. et al., (1980) Crystallographicrefinement and atomic models of the intact immunoglobulin molecule Koland its antigen-binding fragment at 3.0A and 1.9A resolution, J. Mol.Biol. 141:369. The humanized light chain was derived from the humanBence-Jones protein REI, (Epp, O. et al, (1974) Crystal and molecularstructure of a dimer composed of the vandible portion of the Bence-Jonesprotein REI, Eur. J. Biochem. 45:513), with some framework region (FR)changes. The modifications were made to make the VK domain more typicalof human subgroup I, and included replacement of Thr39, Leu104, Gln105and Thr107 with Lys39, Val104, Glu105 and Lys107. In addition, Met4 waschanged to Leu4 to accommodate a PvuII restriction site.

DNA containing the NEWM V_(H) and REI V_(κ) FRs with irrelevant CDRswere cloned into the vectors M13VHPCR1 and M13VKPCR1 (Favaloro et al.Supra). DNA encoding the KOL V_(H) was constructed by a series ofsequential PCRs, using oligodeoxyribonucleotides encoding KOL FR aminoacids and irrelevant CDRs. The constructs were then cloned intoM13VHPCR1.

Oligodeoxyribonucleotides were synthesized to encode the mAB M22 CDRswhich were flanked by nucleotides corresponding to the human FRs. Forthe humanized V_(H) based on NEWM, the primers included murine FR aminoacids Phe27, IIe28 and Arg71 since these were likely to influenceantigen binding, (Chothia, C. and A. M. Lesk (1987), Canonicalstructures for the hypervariable regions of immunoglobulins, J. Mol.Biol., 196:901; Tramontano, A. et al., (1990), Framework residue 71 is amajor determinant of the position and conformation of the secondhypervariable region in V_(H) domains of immunoglobulins, J. Mol. Biol.,215:175). For the humanized V_(κ), murine amino acid Phe7l was similarlyincluded as a residue capable of affecting affinity, (Foote, J. and G.Winter, (1992), Antibody framework residues affecting the conformationof the hypervariable loops, J. Mol. Biol. 224:487. No murine FR residueswere included in the KOL V_(H). Oligodeoxyribonucleotides were 5′-phosphorylated and with the M13 universal forward primer annealed to thehuman V region genes cloned in M13 in reactions containing M13 ssDNAtemplate. The DNA was extended and ligated with 2.5 U T7 DNA polymerase(United States Biochemicals, Cleveland, Ohio) and 0.5 U T4 DNA ligase(Gibco BRL, Grand Island, N.Y.). The mutated strand was preferentiallyamplified from the extension/ligation mixture using M13 reversesequencing primer with 1 U Vent DNA polymerase (New England Biolabs,Beverly, Mass.) and was then amplified by PCR using both M13 forward andreverse primers. Product DNA was cut with BamH1 and HindIII, cloned intoM13 and triple CDR-grafted mutants identified by DNA sequencing.

M13 clones containing the humanized V regions were sequenced in theirentirety to ensure the absence of spurious mutations. RF DNA from theconfirmed clones was digested with HindIII and BamHI, cloned into pSVgptor pSVhyg and human IgG1 or human kappa constant regions added exactlyas described for the construction of the chimeric antibody genes.

Expression and Purification of Recombinant mabs

Heavy (5 μg) and light (10 μg) chain expression vectors were digestedwith PvuI, ethanol precipitated and dissolved in 50 μl water. NSO cells(1-2×10⁷) were harvested by centrifugation, resuspended in 0.5 ml DMEMand mixed with the DNA in a 0.4 cm electroporation cuvette. After 5 min.on ice the cells were given a single pulse of 170 V, 960 μF (GenePulser,Bio-Rad, Melville, N.Y.) and incubated further for 15 min. on ice. Thecells were allowed to recover in DMEM for 24-48 hours. The medium wasthen made selective by the addition of mycophenolic acid (0.8 ug/ml) andxanthine (250 μg/ml). Aliquots of 200 μl were distributed into 96-wellplates. After a further 10-12 days, cells from the wells containing thehighest levels of antibody measured by ELISA were selected and cloned bylimiting dilution.

Antibodies were purified from overgrown cultures by protein A affinitychromatography (Boehringer Mannheim, Lewes, U.K.) Concentrations weredetermined by measuring A_(280nm) and confirmed by ELISA and SDS-PAGE.

ELISA for Measurement of Antibody Binding

The wells of a microtiter plate were coated with goat anti-human IgMantibodies (Sera-Lab, Crawley Down, U.K.) in 50 mM bicarbonate buffer,pH 9.6. The plate was blocked with 1% BSA and followed by the additionof a soluble fusion protein consisting of the extracellular domain ofhuman FcγRI and human IgM heavy chain (sFcγRI-μ) obtained fromtransiently transfected COS cells (the expression vector was kindlyprovided by Dr. Brian Seed, Massachusetts General Hospital, Boston,Mass.). Recombinant 22 or control mAbs were then added in the presenceof excess (2.2 μg/well) human IgG1 antibodies (Sigma, St. Louis, Mo.)that contained λ light chains to block the non-specific binding of thetest mAbs via their Fc portion. Bound 22 mAbs were detected withperoxidase-labeled goat anti-human kappa chain antibodies (Sera-Lab,Crawley Down, U.K.) and o-phenylenediamine.

Fluoresceination of Antibodies

The pH of mAb solution was adjusted to 9.3 by the addition of 0.1MNa₂CO3. Fluorescein iso-thiocyanate (FITC) (Sigma, St. Louis, Mo.) wasdissolved in DMSO at a concentration of 2 mg/ml. Forty μg of FITC wasadded for each milligram of mAb and incubated for two hours at roomtemperature. The fluoresceinated mAb was separated from the free FITC byG-25 chromatography.

Preparation of Blood Cells

Buffy coats were prepared from heparinized whole venous blood. Wholeblood was diluted with RPMI containing 5% dextran at a ratio of 2.5:1(v/v). The erythrocytes were allowed to sediment for 45 minutes on ice,then the cells in the supernatant were transferred to a new tube andpelleted by centrifugation. The residual erythrocytes were removed byhypotonic lysis. The remaining lymphocytes, monocytes and neutrophilswere kept on ice until use in binding assays. For some experiments,neutrophils were separated from mononuclear cells by ficoll hypaque(Pharmacia, Piscataway, N.J.) gradient separation. To up-regulate FcγRI,neutrophils and mononuclear cells were treated with cytokines. Culturesof mononuclear cells were incubated at 37° C., 5% CO₂ for 48 hours inteflon dishes at 4×10⁶ cells/ml of RPMI containing 2.5% normal humanserum type AB (Sigma, St. Louis, Mo.) and 500 IRU/ml IFN-γ (R&D Systems,Minneapolis, Minn.). Neutrophils were cultured for 48 hours (37° C., 5%CO₂) in AIM V media (Gibco, Grand Island, N.Y.) with 50 ng/ml G-CSF(Kindly provided by R. Repp, U. of Erlanger, Germany) and 500 IRU/mlIFN-γ.

Flow Cytometry

Cell binding assays were performed using 96-well microtiter plates aspreviously described, (Guyre, P.M. et al., Monoclonal antibodies thatbind to distinct epitopes on FcγR are able to trigger receptor function.J. Immunol., 143:1650). Briefly, cells were washed in PBS, pH 7.4containing 2mg/ml BSA and 0.05% NaN₃ (PBA), and adjusted to 2.0×10⁷cells/ml with PBA. FITC-labeled and unconjugated antibodies wereprepared in PBA. Cells (25 μl), antibody (25 μl) and human serum (25μl), or human IgG (10 mg/ml, Sigma, St. Louis, Mo.) (25 μl), or PBA (25μl) were added to the microtiter plate, and left on ice for 45-60minutes. Unbound antibody was removed from the wells by washing thecells 3 times with PBA. The cells were fixed with 1% paraformaldehyde.Cell associated fluorescence was analyzed on a Becton Dickinson FACScan.

BsAb Coupling Procedure

BsAb were constructed using the method of Glennie et al, (Glennie, M.J.et al., (1987), Preparation and performance of bispecific F(ab′gamma)²,antibody containing thioether-linked Fab′ gamma fragments, J. Immunol.,139:2367). mAbs 22 (both murine and humanized) and 520C9 (anti-HER2/neu)antibodies were produced by in vitro cultivation of the respectivehybridoma cells. The antibodies were separately digested with pepsin toF(ab′)₂, and subsequently reduced to Fab′by addition of 10 nMmercaptoethanolamine (MEA) for 30 minutes at 30° C. The Fab′ fragmentswere applied to a Sephadex G-25 column equilibrated in 50 mM Na Acetate,0.5rnM EDTA, pH 5.3 (4° C.). Ortho-phenylenedimaleimide (o-PDM, 12mM)dissolved in dimethyl formamide and chilled in a methanol/ice bath wasadded (one half volume) to the murine 22 Fab′in the case of M 22x520C9,and to 520C9 Fab′ in the case of H 22x520C9 and incubated for 30 minuteson ice. The Fab′-maleimide was then separated from free o-PDM onSephadex G-25 equilibrated in 50 mM Na Acetate, 0.5 mM EDTA, pH 5.3 (4°C.). For preparation of the BsAbs, the M22 Fab′-maleimide was added tothe 520C9 Fab′ or the 520C9 Fab′-maleimide was added to H22 Fab′ at a1:1 molar ratio. The reactants were concentrated under nitrogen to thestarting volume using a Diaflo membrane in an Amicon chamber (all at 4°C.). After 18 hours the pH was adjusted to 8.0 with 1M Tris-HCl , pH8.0. The mixture was then reduced with 10 mM MEA (30 minutes, 30° C.)and alkylated with 25 mM iodoacetamide. The bispecific F(ab′)₂ wasseparated from unreacted Fab's and other products by a Superdex 200(Pharmacia, Piscataway, N.J.) column equilibrated in PBS.

Antibody Dependent Cellular Cytotoxicity (ADCC)

The HER2/neu over-expressing human breast carcinoma cells, SKBR-3, wereused as targets for lysis by cytokine activated neutrophils (seepreparation of blood cells). Targets were labeled with 100 μCi of ⁵¹Crfor 1 hour prior to combining with neutrophils and antibodies in aU-bottom microtiter plate. After incubation for 5 hours at 37° C.supernatants were collected and analyzed for radioactivity. Cytotoxicitywas calculated by the formula: % lysis=(experimental CPM−target leakCPM/detergent lysis CPM−target leak CPM)×100%. Specific lysis=% lysiswith antibody−% lysis without antibody. Assays were performed intriplicate.

Superoxide Induction

U937 cells were used for measuring the ability of H22 to trigger asuperoxide burst via FcγRI, (Pfefferkorn, L. C. and G. R. Yeaman (1994),Association of IgA-Fc receptors (FcxR) with Fcε RIγ2 subunits in U937cells, J. Immunol. 153:3228; Hallet, H. B. and A. K. Campbell (1983).Two distinct mechanisms for stimulating of oxygen—radical production inpolymorphonuclear leucocytes, Biochem J. 216:459). U937 cells werecultured for five days in RPMI-1640 (Gibco, Grand Island, N.Y.) with 10%FBS (Hyclone, Logan, Utah) in the presence of 100 U/ml IFN-γ (Genentech,S. San Francisco, Calif.) to induce differentiation and increasedexpression of FcγRI. On the day of the experiment, these differentiatedcells were incubated for 20 minutes in fresh RPMI-1640 with 10% FBS at37° C. The cells were then pelleted and resuspended at a concentrationof 3×10⁶ cells/ml in PBS supplemented with 1 mM CaCl₂, 1 mM MgCl₂, 11 mMglucose, and 100 μg/ml BSA (Sigma, St. Louis, Mo.). To trigger therelease of superoxide, 100 μl of cells were added to 100 μl of areaction solution containing 0.1 mM luminol (Sigma, St. Louis, Mo.), 0.5mM sodium vanadate (Sigma, St. Louis, Mo.), and either mAb M22, H22, or197 and placed in the luminometer at 22° C. Measurements of thespontaneous production of superoxide were made every 30 to 40 secondsstarting immediately following the addition of the cells to the reactionsolution in the luminometer. To compare the superoxide triggered bycrosslinking FcγRI with M22, H22 or 197, each mAb was used at aconcentration of 10 μg/ml. The production of superoxide in mV/sec wasmonitored for 20 minutes. MAb M22, M32.2 and 197 were added at variousconcentrations to establish the dose-responsiveness of superoxideproduction.

Results

Murine Ig VRegion Genes

Ig V region cDNAs were prepared from M22 hybridoma RNA using primersspecific for murine heavy and kappa constant regions and were amplifiedby PCR with the additional use of a series of primers based on sequencesof known signal and/or 5′sequences of mature V regions. PCR products ofthe expected sizes for V_(H) and V_(κ) were obtained using theSH2BACK/CG1FOR and VK7BACK/CK2FOR primer combinations. Amplified DNA wasdigested with appropriate restriction enzymes, cloned into M13 and thesequence in both directions determined from at least 24 independentclones. The deduced amino acid sequences are shown in SEQ. ID Nos. 29and 30. The 4 N-terminal residues of V_(κ) are encoded by the VKBACKprimer.

The M22 V_(H) and V_(κ) are members of murine heavy chain subgroup IIIDand kappa subgroup I, (Kabat, E.A. et al., (1991), Sequences of Proteinsof Immunological Interest, 5th Ed., U.S. Department of Health and HumanServices), respectively. Apart from the residue at L97, the amino acidsequence of the M22 V_(κ) is identical to that from the murine anti-IgGmAb A17 (Shlomchik, M. et al., Variable region sequences of murine IgManti-IgG monoclonal autoantibodies (rheumatoid factors). II Comparisonof hybridonias derived bylipopolysaccharide stimulation and secondaryprotein immunization, J. Exp. Med. 165:970).

Humanized mAbs and Initial Characterization of their Binding M22 V_(H)FR showed greater homology (79%) to KOL (human subgroup III) than toNEWM (57%) (human subgroup II). To see how this difference might affectbinding, heavy chains were constructed based either on NEWM V_(H)including the murine residues Phe27, Ile28 and Arg71, or on KOL V_(H)with no murine FR amino acids. Both humanized V_(H) were partnered withthe same REI-derived humanized light chain.

The affinity of the humanized mAbs was initially assessed by ELISAmeasuring the binding to FcγRI/IgM heavy chain fusion protein. The datashowed that the KOL V_(H)/REI V_(κ) mAb had the same binding as thechimeric mAb whereas the NEWM V_(H)/REI V_(κ) mAb exhibited anapproximate 5- fold lower affinity. The low binding of a nonspecifichuman IgG1 mAb showed that >95% of binding of the humanized mabs was viathe Fv portion rather than through the Fc domain.

While additional changes to the NEWM FR would be expected to restorebinding affinity these could create novel epitopes which might provokean unwanted immunological response. The KOL V_(H)/REI V_(κ) mAb,designated H22, was therefore chosen for further examination of itsbinding characteristics.

Functional Characterization of mA bH22

A series of binding experiments were performed to establish thespecificity and isotype of the H22 antibody. Peripheral blood leukocytesstained with fluorescein-conjugated M22 or H22 demonstrated specificbinding to monocytes with approximately 10⁴ binding sites per cell. Incontrast, lymphocytes or unstimulated neutrophils had little or nospecific binding (Table 1):

TABLE 1 Specific Binding of H22 to Monocytes Antibody MonocytesLymphocytes PMNs M22 10,000^(a) <1000 <1000 H22 10,500 <1000 <1000^(a)Antibody sites per cell, average of duplicates

To demonstrate that the H22 binds to FcγRI at the same site as M22 andthat it also binds as a ligand at the Fc binding domain, competitionexperiments with two anti-FcγRI murine mAb (M22 and M32.2) and a humanIgG1 mAb were performed. Unconjugated H22 and M22 competed equivalentlyfor either the binding of fluoresceinated M22 or fluoresceinated H22 inthe presence of excess human IgG which saturated the Fc binding sites onFcγRI. As expected, the anti-FcγRI antibody M32.2 which binds to adifferent site on FcγRI than M22 (Guyre, P. M. et al., J. Immunol.143:1650) was also unable to compete with the M22-FITC. In addition, theinhibition of H22-FITC by H22 and not by an irrelevant human IgG1 mAbconfirmed the specificity of FcγRI binding via the V regions of H22.

H22, but not M22, was able to compete for Fc mediated binding to FcγRIby a fluorosceinated human IgG1. This experiment demonstrated that theFc portion of H22 but not M22 bound to the Fc binding domain of FcγRI.This is consistent with the ability of the Fc portion of human IgG1antibodies, but not murine IgG1, to bind FcγRI with high affinity.

Since the humanization of M22 was primarily to increase itsimmunotherapeutic potential, the binding activity of H22 to monocytesand cytokine-activated neutrophlils was determined in the presence ofhuman serum. H22-FITC bound with similar affinity to FcγRI on monocytesin the presence or absence of human serum. In contrast, the Fc-mediatedbinding of an irrelevant human IgG-FITC was completely inhibited byhuman serum. Likewise, H22-FITC bound with similar affinity toIFN-γ-treated neutrophils in the absence and in the presence of humanserum. Collectively, the data demonstrated that H22 binds both via its Vregions to a site distinct from the Fc binding domain and via its Fcregion to the ligand binding domain of FcγRI. The former bindingactivity effectively overcomes antibody blockade of human IgG1.

Functional Activity of H22 BsAb

The foremost application of anti-FcγRI antibodies for immunotherapy isthe development of BsAbs which link FcγRI-bearing effector cells to atumor cell, a virus, or a virally-infected cell. Such BsAb have beendeveloped with M22; therefore, a comparison was made of the ability ofthe M22 anti-tumor BsAb (520C9xM22) and a corresponding H22 BsAb(520C9xH22) to mediate cytotoxicity. These BsAbs consisted of H22 or M22Fab′ chemically conjugated to the Fab′ of an anti-HER2/neu antibody(520C9), and thus were specific for the effector cell trigger moleculeFcγRI and the tumor antigen.

Comparison of M22-derived and H22-derived BsAbs was done by ADCC assays.M22- and H22-derived BsAbs mediated the killing of HER2/neuoverexpressing SKBR-3 cells. Both the murine and humanized BsAbsexhibited similar levels of lysis of antigen bearing target cells. Inaddition, both BsAb retained ADCC activity in the presence of humanserum, while excess M22 F(ab′)₂ resulted in complete inhibition ofkilling. Taken together these results show that the H22 BsAb-inducedlysis is mediated through the M22 epitope and that the ADCC is FcγRIspecific.

Finally, the ability of H22 and M22 to stimulate superoxide productionby the monocyte-like cell line U937 was evaluated. M22, which binds tothe FcγRI only by its V regions, induced a very low level oxygen burst,presumably because it is unable to cross-link the receptor efficiently.However, H22, which can cross-link FcγRI by binding as a ligand via itsFc domain and, additionally, as an antibody via its Fv, induced a moresubstantial release of superoxide.

Example 2

Generation of a Functional H22-Epidermal Growth Factor Fusion Protein

Materials and Methods

Expression Vectors and Cloning

Expression vectors for the genomic clones of the heavy (pSVgpt) andlight (pSVhyg) chains of H22 are as described in International PatentApplication Publication Number: WO 94/10332 entitled, HumanizedAntibodies to Fc Receptors for Immunoglobulin G on Human MononuclearPhagocytes. For the Fab-ligand fusion construct, it was unnecessary toalter the light chain. For the heavy chain, however, the CH2 and CH3domains had to be removed and replaced with the coding sequences of theligands. The heavy chain vector contains two BamHI sites, one in theintron between V_(H) and CH1, and the other just downstream of CH3.Using the BamHI restriction sites, DNA encoding the constant domainswere replaced by a truncated version encoding only CH1 and most of thehinge. To do this, the polymerase chain reaction (PCR) was utilized toengineer the new C-terminus of the heavy chain fragment with thealterations shown in FIG. 1.

The construct shown in FIG. 1 [C], consisting of a translationtermination codon downstream of the cloning restriction sites, XhoI andNotI, and upstream of a BamHI site which was used to clone the new PCRgenerated CHI fragment downstream of VH, was used to generate the fusionprotein constructs. The cloning sites, which are located downstream ofmost of the hinge in order to retain flexibility between the Fd andligand domains, was used to insert DNA encoding EGF or other ligands.Also, the single Cys residue has been retained from the previousconstruct to allow conjugation for the formation of dimeric molecules.

DNA encoding the ligands were amplified by PCR to have a XhoI site onthe N-terminus and a NotI site on the C-terminus of the coding region,and then inserted in the proper reading frame into the same sites of thenewly engineered H22 heavy chain truncated fragment described above.cDNA encoding epidermal growth factor (EGF) was obtained from the ATCC(#59957). Only DNA encoding the 53 amino acid residues of mature EGF outof the approximately 1200 residue precursor was cloned beginning withAsn 971 and ending with Arg 1023 (Bell, G. I., Fong, N. M., Stempien, M.M., Wormsted, MA., Caput, D., Ku. L., Urdea, M. S., Rall, L. B. &Sanchez-Pescador, R. Human Epidermal Growth Factor Precurser: cDNASequence, Expression In Vitro and Gene Organization. Nucl. Acids Res.14: 8427-8446,1986.).

Expression

The murine myeloma NSO (ECACC 85110503) is a non-Ig synthesizing lineand was used for expression of the fusion proteins. The final expressionvector, a pSVgpt construct with DNA encoding H22 Fd fused in frame toEGF (shown in FIG. 2) was transfected by electroporation using a BioRadGene Pulser to NSO which had been previously transfected with the pSVhygconstruct containing DNA encoding H22 light chain. These polypeptideswere expressed by an Ig promoter and Ig enhancer present in the vectors,and secreted by the mAb 22 heavy chain signal peptide located on theN-terminus of the constructs. One or two days after transfection,mycophenolic acid and xanthine were added to the media to select forcells that took up the DNA. Individual growing colonies were isolatedand subcloned after binding activity was demonstrated by ELISA.

Purification

Cells expressing the H22-EGF fusion protein were subcloned and expanded.The fusion protein-expressing clone was expanded and grown in spinnercultures and the supernatant was clarified and concentrated. Small scalepurification was performed by affinity chromatography on an anti-humankappa chain affinity column (Sterogene. Carlsbad, Calif.). The purifiedprotein was analyzed by SDS-PAGE on a 5-15% acrylamide gradient gelunder nonreducing conditions. FIG. 3 is a schematic representation ofthe generation of anti-Fc receptor-ligand fusion proteins.

Bispecific flow cytometry

To show that the fusion protein is capable of binding both FcγRI andEGFR simultaneously, a flow cytometric assay has been developed (FIG.4). In this assay different concentrations of H22-EGF fusion protein orthe bispecific antibody, BsAb H447 (H22xH425, a humanized version of themurine monoclonal antibody M425, which binds EGFR at the ligand bindingsite (E. Merck) was incubated with A43 1 cells, a cell line whichexpresses the EGF receptor (EGFR) (ATCC, Rockville, Md.). After washing,a supernatant containing a fusion protein consisting of theextracellular domain of FcγRI and the Fc portion of human IgM was added.Finally, a Phycoerythrin (PE)-labeled mAb (32.2), that binds Fcγ RI at asite that is distinct from that bound by mAb 22, was added. The cellswere then analyzed by FACSCAN. Alternatively, binding to EGFR wasblocked by excess (100 μg/ml) whole murine mAb 425 (E. Merck), andbinding of bsAb or fusion protein was detected by PE-labeled anti-humanIgG.

ADCC

ADCC mediated by the fusion protein was determined using a ⁵¹Cr killingassay. The EGFR overexpressing cell line, A43 1, was used as targets forlysis by human monocytes cultured in γ-interferon (IFN-γ) for 24 hours.Targets were labeled with 100 μCi of 51 Cr for 1 hour prior to combiningwith effector cells and antibodies in a U-bottom microtiter plate. Afterincubation for 5 hours at 37° C. supernatants were collected andanalyzed for radioactivity. Cytotoxicity was calculated by the formula:% lysis=(experimental CPM−target leak CPM/detergent lysis CPM−targetleak CPM)×100%. Specific lysis=% lysis with antibody−% lysis withoutantibody. The ability of the fusion protein to mediate ADCC was comparedwith that of the respective BsAb. The assay was also performed in thepresence of 25% human serum to demonstrate that IgG or other factorsfound in human serum will not inhibit fusion protein-mediated ADCC.

Results

Purification

NSO cells expressing the H22 kappa chain were transfected with theH22-EGF heavy chain construct and clones selected for resistance tomycophenolic acid and xanthine were expanded and the fusion protein wasaffinity-purified from the supernatant on an anti-human kappa column(Sterogene, Carlsbad, Calif.). The purified protein was analysed bySDS-PAGE. The purified protein migrated at an apparent molecular weightof 50-55 kDa, indicating that the fusion protein is expressed as amonomer, not a disulfide-linked dimer. In addition, a band was seen atan apparent molecular weight of 25 kDa and is probably free light chain.

Binding Specificity

To demonstrate that the fusion protein could bind FcγRI and EGFRsimultaneously a bispecific FACS assay was devised. FIG. 5 shows thatboth the chemically-linked, fully-humanized BsAb H447 (H22(anti-FcγRI)xH425), which was made as described in the following Example3, and the H22-EGF fusion protein bound EGFR on A431 cells and solubleFcγRI simultaneously in a dose-dependent fashion.

The EGFR-specificity of the fusion protein was demonstrated by theability of the murine mAb, M425, which binds EGFR at the ligand bindingsite, to inhibit fusion protein or H22xH425 binding. Variousconcentrations of either the BsAb H447, or of the H22-EGF fusion proteinwere incubated with A431 cells in either the presence or absence of anexcess of M425. FIG. 6 shows that binding of both the BsAb and thefusion protein were inhibited by M425, demonstrating the specificity ofthe fusion protein for EGFR.

ADCC

The ability of the fusion protein to mediate ADCC was analyzed usingA431 cells as targets. Human monocytes cultured for 24 hours in thepresence of IFN-γ were used as effector cells. FIG. 7 demonstrates thewhole antibody, H425, the BsAb H447 (H22xH425) and the fusion proteinmediated dose-dependent lysis of A431 cells. FIG. 8 demonstrates thatwhile ADCC mediated by the whole antibody is inhibited by 25% humanserum (25%HS), ADCC mediated by the fusion protein was not inhibited byhuman serum and, in this particular experiment, fusion protein-mediatedADCC was enhanced by human serum. These results support the clinicalutility of these molecules by demonstrating that the fusion protein wascapable of killing EGFR-overexpressing cells, even in the presence ofFcγ RI-expressing effector cells as would be present in vivo.

Growth Inhibitory Properties of H22-EGF Fusion Proteins

Although EGF acts to stimulate growth of normal cells that expressreceptors for it, EGF also can act to inhibit growth of tumor cells thatover-express EGF-R (Barnes, D. W. (1982) J. Cell Biol. 93:1, MacLeod, C.L. et al. (1986) J. Cell. Physiol. 27:175). The ability of EGF and theH22-EGF fusion protein to inhibit the growth of A431 cells was examinedas follows.

2×10⁴ A431 cells were added to six well plates in complete media aloneor in media containing various concentration of either EGF, H22-EGF, theFab fragment of H22, or the F(ab′)₂ fragment of H425. Viable cells werecounted after seven days using a hemocytometer. The analyses wereperformed in duplicate and reported as means +/− standard deviations.

The results are presented in FIG. 9. These results indicate that EGF and22-EGF significantly inhibited cell growth in a dose dependent fashion.On the contrary, he F(ab′)₂ fragment of H425 which had some inhibitoryactivity only at high concentrations and the Fab fragment of H22 had nogrowth inhibiting activity.

Thus, H22-EGF is able to bind to both FcγRI and EGF simultaneously,indicating that the molecule had folded properly and had maintained theflexibility required to bind both receptors at the same time.Furthermore, H22-EGF inhibited proliferation of the EGF-R expressingtumor cell line, A43 1, indicating that, similar to EGF, the H22-EGFfusion protein is capable of signaling through the EGF-R. H22-EGF alsomediates potent killing of A431 cells in the presence of FcγRIexpressing effector cells. Thus, H22-EGF mediates both cytotoxic andcytostatic effects on EGF-R expressing cells. Administration of H22-EGFto a subject having a tumor will result in recruitment of the body'snatural cytotoxic effector cells to mediate killing of the tumor cellsby potentially three different modes-cytotoxicity, growth inhibition,and phagocytosis. Furthermore, in addition to cell mediated cytotoxicityof the tumor cells, the effector cells recruited by H22-EGF may alsofurther augment anti-tumor immunity by secreting inflammatory cytokinesand/or by processing and presenting tumor antigens to tumor specific Tcells.

Example 3

H22-Heregulin (H22-gp30) Fusion Protein Mediates Tumor Cell Killing

Heregulin (HRG) is a ligand for the HER3 and HER4 molecules. Both ofthese receptors may form heterodimers with HER2, a molecule which isoverexpressed in some breast cancer cells. The affinity of HRG for HER3and HER4 increases significantly when these molecules from heterodimerswith HER2. This example demonstrates that a bispecific molecule,comprising heregulin and a binding specificity for the FcγRI inhibitsgrowth of a tumor cell line and mediates fusion protein dependentcytotoxicity of these cells in the presence of FcγRI-bearing cytotoxiceffector cells.

The H22-heregulin fusion protein was constructed in the same manner asthe H22-EGF fusion protein described in Example 2. Briefly, genornic DNAencoding the Fd fragment of humanized anti-FcγRI mAb, H22, was fused tocDNA encoding the EGF domain of the β2 form of HRG. The amino acidsequence of the H22-HRG fusion protein (SEQ ID NO: 4) is shown in FIG.10. This fusion protein comprises amino acids 171-239 of the heregulinβ2 shown in U.S. Pat. No. 5,367,060. Other portions of heregulin β2, aswell as portions of other heregulin molecules, such as those disclosedin U.S. Pat. No. 5,367,060 can also be used. The resulting H22Fd-HRGexpressing vector was transfected into a myeloma cell line previouslytransfected with a vector containing DNA encoding the H22 kappa lightchain. The resultant fusion protein was expressed predominantly as amonomer, even though the protein contains a free Cys residue in thehinge region of the H22 Fab component. Flow cytometry showed that thisfusion protein was able to bind to the HER2 overexpressing tumor cellline, SKBR-3, as well as to FcγR-expressing cells.

To test the biological activity of the H22Fd-HRG fusion protein,supernatant from the myeloma cells expressing this fusion protein wasdiluted three fold or thirty fold and added to PC-3 cells or SKBR-3tumor cells expressing HER2, HER3, and HER4 in the presence ofIFN-treated monocytes at a ratio of 100:1 monocytes to target tumorcells. The monocytes were treated with IFN-γ and the target cells werelabeled with ⁵¹Cr as described in Example 2. The % of specicific lysiswas calculated as indicated in Example 2. The results are presented inFIG. 11. The results indicate that about 45% of SKBR3 cells and up toabout 49% of PC-3 cells are lysed upon incubation of the cells with thesupernatant diluted 3 fold.

This fusion protein inhibits growth of SKBR-3 tumor cells and mediatesfusion protein dependent cytotoxicity of these cells in the presence ofFcγRI-bearing cytotoxic effector cells. Thus, the results of thisexample show that an anti-FcγRI-heregulin fusion protein can mediateanti-tumor cytotoxic activities under physiologic conditions andindicate that such a fusion protein will have therapeutic utility in thetreatment of various cancers.

Example 4

H22-Bombesin Fusion Protein Mediates Tumor Cell Killing

The H22-bombesin fusion protein was constructed similarly to the H22-EGFfusion protein described above. However, since bombesin is a shortpeptide (14 amino acid residues), instead of amplifying cDNA encodingbombesin using PCR technology, DNA oligomers encoding the sense andanti-sense strands of bombesin were hybridized to create the codingregion. The amino acid sequence of the bombesin peptide fused to thecarboxyl end of the heavy chain of the H22 antibody is the following:

—Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-Gly (SEQ ID NO: 5)which corresponds to amino acids 2-14 of bombesin (Anastasi et al.(1971) Experientia27:166) and contains an additional glycine residue atthe carboxyl end of the peptide. The oligomers had overlapping ends thatdid not hybridize but instead created sticky ends for a XhoI site on theN-terminus and a NotI site on the C-terminus such that it could becloned into the H22 heavy chain expression vector described above.

The biological activity of the H22-bombesin fusion protein on tumor cellkilling was investigated as described above for the H22-EGF andH22-heregulin fusion proteins. Briefly, PC-3 tumor cells bearingbombesin receptors were labeled with ⁵Cr and incubated with monocytesand various concentrations of H22-fusion protein, and fusion proteindependent lysis was determined as described above. The results, shown inFIG. 12, indicate that the target cells are lysed and that the level oftarget cell lysis increases proportionally with the amount of fusionprotein added to the assay.

Fusion proteins having H22 as one binding entity and CD4 (AIDSRepository) or gp120 (AIDS Repository) as a second binding entity werealso produced.

Example 5

Production of Bispecific Antibodies From Modified Humanized AntibodyFragments

Materials and Methods

Expression Vectors and Cloning

Expression vectors for the genomic clones of the heavy (pSVgpt) andlight (pSVhyg) chains of H22 were as described in International PatentApplication Publication Number: WO 94/10332 entitled, HumanizedAntibodies to Fc Receptors for Immunoglobulin G on Human MononuclearPhagocytes. For the Fab′ construct, it was unnecessary to alter thelight chain. For the heavy chain, however, the CH2 and CH3 domains hadto be removed and replaced with a termination codon. The heavy chainvector contains two BamHlI sites, one in the intron between V_(H) andCH1, and the other just downstream of CH3. Using the BamHI restrictionsites, DNA encoding the constant domains were replaced by a truncatedversion encoding only CH1 and most of the hinge. To do this, Thepolymerase chain reaction (PCR) was utilized to engineer the newC-terminus of the heavy chain fragment with the alterations shown inFIG. 1. FIG. 1 [B] shows the alterations for generation of a truncatedsingle-sulfhydryl version.

Expression

The murine myeloma NSO (ECACC 85110503) is a non-Ig synthesizing lineand was used for expression of the modified H22 antibody. The finalexpression vector, a pSVgpt construct with DNA encoding H22 Fd wascotransfected with the pSVhyg construct containing DNA encoding H22light chain by electroporation using a BioRad Gene Pulser. Thesepolypeptides were expressed by an Ig promoter and Ig enhancer present inthe vectors, and secreted by the mAb 22 heavy chain signal peptidelocated on the N-terminus of the constructs. One or two days aftertransfection, mycophenolic acid and xanthine were added to the media toselect for cells that took up the DNA. Individual growing colonies wereisolated and subeloned after FcγRI binding activity was demonstrated.

Purification

The single sulfhydryl form of the H22 antibody and the whole H425(anti-EGFR) antibody were produced by in vitro cultivation of therespective transfected NSO cells. The H425 was purified by protein Aaffinity chromatography. The single sulfydryl form of the antibody H22was purified by ion exchange chromatography using Q-Sepharose followedby SP-Sepharose (Pharmacia, Piscataway, NJ). The purity of the singlesulfhydryl form of the H22 antibody was assessed by SDS-PAGE.

Generation of Bispecific Antibody (BsAb)

BsAb was constructed using the method of Glennie et al. (Glennie, M. J.et al., (1987), Preparation and performance of bispecific F(ab′ gamma)²,antibody containing thioether-linked Fab′ gamma fragments, J. Immunol.,139:2367). The F(ab′)₂ of H425 was generated by limited pepsinproteolysis in 0.1M citrate buffer, pH 3.5 and the F(ab′)₂ purified byion exchange chromatography. The mabs were reduced by addition of 20 mMmercaptoethanolamine (MEA) for 30 minutes at 30° C. The Fab′fragmentswere applied to a Sephadex G-25 column equilibrated in 50 mM sodiumacetate, 0.5 mM EDTA, pH 5.3 (4° C.). Ortho-phenylenedimaleimide (o-PDM,12 mM) dissolved in dimethyl formamide and chilled in a methanol/icebath was added (one half volume) to the H22 Fab′ and incubated for 30minutes on ice. The Fab′-maleimide was then separated from free o-PDM onSephadex G-25 equilibrated in 50 mM Na Acetate, 0.5 mM EDTA, pH 5.3 (4°C.). For preparation of the BsAbs, the H22 Fab′-maleimide was added tothe H425 Fab′ at a 1.2:1 molar ratio. The reactants were concentratedunder nitrogen to the starting volume using a Diaflo membrane in anAmicon chamber (all at 4° C.). After 18 hours the pH was adjusted to 8.0with 1M Tris-HCl, pH 8.0. The mixture was then reduced with 10 mM MEA(30 minutes, 30° C.) and alkylated with 25 mM iodoacetamide. Thebispecific F(ab′)₂ was separated from unreacted Fab's and other productsby a Superdex 200 (Pharmacia, Piscataway, N.J.) column equilibrated inPBS.

Bispecific Flow Cytometry

To show that BsAb generated by the o-PDM method as well as thatgenerated by the DTNB method are capable of binding both FcγRI and EGFRsimultaneously, a flow cytometric assay has been developed (FIG. 13). Inthis assay different concentrations of the two BsAbs were incubated withA431 cells, a cell line which expresses the EGF receptor (EGFR). Afterwashing, a supernatant containing a fusion protein consisting of theextracellular domain of FcγRI and the Fc portion of human IgM wasincubated with the cells. Finally, the cells were incubated with aFITC-labeled anti-human IgM-specific antibody. The cells were thenanalyzed by FACSCAN.

ADCC

BsAb-mediated ADCC was determined using a ⁵¹Cr killing assay. The EGFRoverexpressing cell line, A43 1, was used as targets for lysis by humanmonocytes cultured in y-interferon for 24 hours. Targets were labeledwith 100 μCi of ⁵¹Cr for 1 hour prior to combining with effector cellsand antibody in a flat-bottomed microtier plate. After incubation for 16hours at 37° C. supernatants were collected and analyzed forradioactivity. Cytotoxicity was calculated by the formula: %lysis=(experimental CPM−target leak CPM/detergent lysis CPM−target leakCPM)×100%. Ab-dependent lysis=% lysis with antibody−% lysis withoutantibody.

Results

Purification

NSO cells were cotransfected with the truncated H22 heavy chainconstruct and the intact kappa chain construct. Clones selected forresistance to mycophenolic acid and xanthine were expanded and theprotein was purified from the supernatant by Q-Sepharose followed bySP-Sepharose ion exchange chromatography. The purified protein wasanalyzed by SDS-PAGE. The purified protein migrated at an apparentmolecular weight of 50 kDa, indicating that the protein is expressed asa monomer, not a disulfide-linked dimer.

Construction and Characterization of a BsAb Composed of SingleSulfhydryl H22 Linked to Fab′ of H425 (anti-EGFR)

A BsAb was constructed where the single sulfhydryl form of H22 waslinked to the Fab′ fragment of H425, a humanized anti-EGFR mAb. The BsAbwas generated using o-PDM as a linker by the method of Glennie et al.(Glennie, M. J. et al., (1987), Preparation and performance ofbispecific F(ab′ gamma)², antibody containing thioether-linked Fab′gamma fragments, J. Immunol., 139:2367). The activity of this BsAb wascompared to one generated by the DTNB method using Fab′ fragments madefrom pepsin digestion and reduction of whole H22. To demonstrate thatthese BsAbs could bind FcγRI and EGFR simultaneously a bispecific FACSassay was devised. FIG. 14 shows that both the o-PDM-linked BsAb and theBsAb made by the DTNB method bound EGFR on A43 1 cells and soluble FcγRIsimultaneously in a dose-dependent fashion.

The ability of the two BsAbs to mediate ADCC was analyzed using A431cells as targets. Human monocytes cultured for 24 hours in the presenceof IFN-γ were used as effector cells. FIG. 15 demonstrates the two BsAbsmediated dose-dependent lysis of A431 cells in a comparable fashion.These results demonstrated that BsAb generated from the truncated,single sulfhydryl form of H22 was capable of killing EGFR-overexpressingcells in the presence of FcγRI-expressing effector cells.

Example 6

Production of Trivalent Antibodies

Materials and Methods

Cell lines and Antibodies. M22, 520C9, H425. SKBR3 and A431

M22 and 520C9 were purified from hybridoma supernatant by ion exchangechromatography (Pharmacia, Piscataway, N.J.) and 520C9 was furtherpurified by protein A affinity chromatography (Phannacia, Piscataway,N.J.). H425 was purified from hybridoma supernatant by protein Aaffinity chromatography (Pharmacia, Piscataway, N.J.). The M22- and520C9- producing murine hybridoma were described previously (Guyre etal., (1989) Monoclonal antibodies that bind to distinct epitopes onFcgRI are able to trigger receptor function, J. Immunol. 143:5,1650-1655; Frankel et al., (1985) Tissue distribution of breastcancer-associated antigens defined by monoclonal antibodies, J. Biol.Response Modifiers, 4:273-286). The murine myeloma NSO (ECACC 85110503)is a non-Ig synthesizing line and was used for the expression of thehumanized mAb, H425 (Kettleborough et al., (1991) Humanization of amouse monoclonal antibody by CDR-grafting: the importance of frameworkresidues on loop conformation, Protein Eng., 4:773). SKBR-3, (ATCC,Rockville, Md.) a human breast carcinoma cell line that overexpressesthe HER2/neu protooncogene, and A431 (ATCC, Rockville, Md.), a humansquamous carcinoma cell line that overexpresses EGFR (ATCC, Rockville,Md.) were cultivated in Iscove's Modified Dulbecco's Medium (IMDM,Gibco, Grand Island, N.Y.).

Neutrophil Preparation

Neutrophils are separated from mononuclear cells by ficoll hypaque(Pharmacia, Piscataway, NJ) gradient separation. To up-regulateF_(cγ)RI, neutrophils are treated with cytokines. Neutrophils arecultured for 24-48 hrs (37° C., 5% CO₂) in AIM V media (Gibco, GrandIsland, N.Y.) containing 2.5% normal human serum type AB (Sigma, St.Louis, Mo.), 50 ng/ml G-CSF (Kindly provided br R. Repp, U. of Erlanger,Germany) and 100 IRU/ml IFN-γ.

Conjugation Method

BsAb were constructed using the method of Glennie et al (Glennie, M. J.et al., (1987), Preparation and performance of bispecific F(ab′ gamma)²,antibody containing thioether-linked Fab′gamma fragments, J. Immunol.,139:2367). mAbs M22, 520C9 (anti-HER2/neu, 33), and H425 (anti-EGFR)antibodies were produced by in vitro cultivation of the respectivehybridoma cells. The F(ab′)2 of each antibody were generated by limitedpepsin proteolysis in 0.1 M citrate buffer, pH 3.5 and the F(ab′)₂purified by ion exchange chromatography. mAbs M22 and H425 were reducedto Fab′ by addition of 20 mM mercaptoethanolamine (MEA) for 30 minutesat 30° C. The Fab′ fragments were applied to a Sephadex G-25 columnequilibrated in 50 mM Na Acetate, 0.5 mM EDTA, pH 5.3 (4° C.).Ortho-phenylenedimaleimide (o-PDM, 12 mM) dissolved in dimethylformamide and chilled in a methanol/ice bath was added (one half volume)to the murine 22 Fab′ and incubated for 30 minutes on ice. TheFab′-maleimide was then separated from free o-PDM on Sephadex G-25equilibrated in 50 mM Na Acetate, 0.5 mM EDTA, pH 5.3 (4° C.). Forpreparation of the BsAbs, the M22 Fab′-maleimide was added to the H425Fab′ at a 1:1 molar ratio. The reactants were concentrated undernitrogen to the starting volume using a Diaflo membrane in an Amiconchamber (all at 4° C.). After 18 hours the pH was adjusted to 8.0 with1M Tris-HCl, pH 8.0. The mixture was then reduced with 10 mM MEA (30minutes, 30° C.) and alkylated with 25 mM iodoacetamide. The bispecificF(ab′)₂ as separated from unreacted Fab's and other products by aSuperdex 200 (Pharmacia, Piscataway, N.J.) column equilibrated inphosphate buffered saline (PBS). The BsAb M22x520C9 was made in asimilar fashion except that 520C9 was used instead of H425.

Trispecific antibody composed of M22xH425x520C9 was made in two stages(FIG. 16). In the first stage, M22 was linked to H425 as described aboveto create the M22xH425 BsAb except that rather than a final reductionand alkylation, the reactants were treated with DTNB to block theremaining free sulfhydryl groups. The bivalent BsAb was purified by gelfiltration on a Superdex 200 column, reduced to F(ab′)₂(SH) and mixed ina 1:1 molar ratio with o-PDM-treated 520C9. The resulting trispecificF(ab)₃ was purified on a Superdex 200 column. The TsAb was analyzed byHPLC size exclusion chromatography using a TSK 3000 column (ToJo Haas,Japan). Using the same procedure as above another TsAb comprising m22Fab′x32.2 Fab′xm22 Fab′has been constructed.

Bispecific Flow Cytometry

The TsAb can bind to EGFR and F_(cγ)RI simultaneously or to HER2/neu andF_(cγ)RI simultaneously. Either A431 cells (high EGFR-expressing cells)or SKBR-3 cells (high HER2/neu-expressing cells) were incubated withvarious concentrations of BsAbs (M22x520C9 or M22xH425) or with theTsAb, M22xH425x520C9. The cells were washed and then incubated with thesoluble FcγRI. Soluble F_(cγ)RI binding was detected with mAb 32.2-FITCwhich binds F_(cγ)RI at a site that is distinct from the 22 bindingsite. The cells were then analyzed by FACSCAN.

ADCC

Either SKBR-3 cells or A431 cells were used as targets for lysis bycytokine activated neutrophils. Targets were labeled with 100 μCi of⁵¹Cr for 1 hour prior to combining with neutrophils and antibodies in aU-bottom microtiter plate. After incubation for 16 hours at 37° C.supernatants were collected and analyzed for radioactivity. Cytotoxicitywas calculated by the formula: % lysis=(experimental CPM−target leakCPM/detergent lysis CPM−target leak CPM)×100%. Specific lysis=% lysiswith antibody−% lysis without antibody. Assays were performed intriplicate.

FcγRLI Modulation Assay

The M22x32.2xM22 BsAb was used for modulation of FcγRI on monocytes inwhole blood. The assay procedure is shown in the enclosed flow chart(see FIG. 23A). FIG. 23B shows that treatment with 10 μg/nL of this BsAbdecreased the FcγRI expression on monocytes to approximately 50% of thelevel prior to BsAb treatment.

Results

Construction and Biochemical Characterization of the TsAb

TsAb was made according to the flow chart depicted in FIG. 16. In thefirst stage of the procedure, M22 was coupled to H425, treated withDTNB, and the resulting bispecific F(ab′)₂ purified by gel filtration.In the second stage, this bispecific F(ab′)₂ was reduced and mixed witho-PDM-treated 520C9 Fab′ resulting in the TsAb, M22xH425x520C9. ThisTsAb is depicted schematically in FIG. 17. In this figure, Fab′-Arepresents M22, Fab′-B represents H425, and Fab′-C represents 520C9.

Binding (Bs FACS)

To demonstrate that the TsAb, M22xH425x520C9, could bind FcγRI andHER2/neu simultaneously a bispecific FACS assay was devised. This assayis depicted schematically in FIG. 18A. FIG. 19 shows that both the TsAbbound HER2/neu on SKBR-3 cells and soluble FcγRI simultaneously in adose-dependent fashion. The BsAb, M22xH425, generated negligible signalin this assay over a wide range of concentrations. To demonstrate thatthe TsAb, M22xH425x520C9, could bind FcγRI and EGFR simultaneously asimilar assay was devised using the EGFR-overexpressing cell line, A431,in the case. This assay is depicted schematically in FIG. 18B. FIG. 20shows that both the TsAb and the BsAb, M22xH425, bound EGFR on A431cells and soluble FcγRI simultaneously in a dose-dependent fashion. TheBsAb, M22x520C9, generated negligible signal in this assay over a widerange of concentrations.

ADCC

The ability of the TsAb to mediate ADCC was analyzed using either SKBR-3or A431 cells as targets. Human neutrophils cultured for 24-48 hours inthe presence of IFN-γ and G-SF were used as effector cells. FIG. 21demonstrates the both the BsAb, M22x20C9, and the TsAb, M22xH425x520C9,mediated lysis of SKBR-3 cells, whereas the sAb, M22xH425, did not. Onthe other hand, FIG. 22 demonstrates the BsAb, M22xH425, and the TsAb,mediated lysis of SKBR-3 cells, whereas the BsAb, M22x520C9, did not.These results demonstrated that the TsAb was capable of killing bothHER2/neu and EGFR-overexpressing cells in the presence ofF_(cγ)RI-expressing effector cells.

The trispecific antibody described above included M22, the murineversion of the anti-FcγRI mAb. Such a trispecific antibody could beconstructed using the single-sulfhydryl form of the humanized anti-FcγRImAb, H22. The only difference being that single-sulfhydryl form issecreted as a F(ab′)₂ fragment of this antibody. The single-sulfhydrylform is purified from culture supernatants utilizing ion exchangechromatography using Q-Sepharose followed by SP-Sepharose (Pharmacia,Piscataway, N.J.). Once the single-sulfhydryl form of H22 is purified,the creation of a trispecific antibody using this reagent would beidentical to that described above using the F(ab′)₂ fragment of M22.

Example 7

Enhanced Antigen Presentation with H22-antigen Fusion Proteins

This example demonstrates that (a) antigenic peptides geneticallygrafted onto the constant region of an anti-FcγRI antibody aresignificantly more efficient in antigen presentation of the antigen andT cell stimulation compared to the antigen alone, and (b) thatantagonistic peptides genetically grafted onto the constant region of ananti-FcγRI are significantly more efficient in inhibiting T cellstimulation compared to the antagonistic peptide alone. Thus, suchfusion proteins will effectively increase the delivery of peptides toantigen presenting cells (APCs) in vivo and will be useful in varioustherapeutic methods.

Materials and Methods

Reagents

AIM V (GIBCO, Grand Island, N.Y.) was used as culture medium. TetanusToxoid (TT) was purchased from ACCURACTE CHEMICAL CO. (Westbury, N.Y.).Sterile and low-endotoxin F(ab′)₂ fragment of mouse anti-FcγRI mAb 22and the bispecific Ab, MDXH210 (consisting of Fab′ of humanized Ab 22chemically linked to Fab′of anti-Her2/neu tumor Ag mAb 520C9) wereprovided by MEDAREX, INC. (Annandale, N.J.). The universal Th epitope ofTT, TT830-844 (QYIKANSKFIGITEL (SEQ ID NO: 6), termed as TT830hereafter) (Valmori D. et al. (1994) J. Immunol. 152:2921-29) and themutant form of this epitope, TT833S (QYISANSKFIGITEL (SEQ ID NO: 9),lysine at position 833 changed into serine) were synthesized andpurified to >95% by PEPTIDOGENIC CO. (Livermore, Calif.). Anotheruniversal Th epitope of TT, TT947-967 (FNNFTVSF WLRVPKVSASHLE (SEQ IDNO: 12), referred to as TT947 hereafter), (>80% pure) (Valmori D. supra)was used as a control peptide in ths study. Commercially available humanIgG for intravenous injection (IVI) was used in blocking experiments.

Cells

The monocytic cell line, U937, which expresses FcγRI, was obtained fromthe ATCC. The method of generating CD4⁺, peptide TT830-specific T cellswas modified from a previously described protocol for TT-specific T celllines (Gosselin E.J. (1992) J. Immunol. 149:3477-81). Briefly,mononuclear cells were isolated from peripheral blood using FicollHypaque. 150×10⁶ mononuclear cells were stimulated in 50 ml of AIM Vmedium with 10 μM TT830. After three days' incubation at 37° C. in a 5%CO₂ incubator, non-attached (mostly non-specific cells) were removed bywashing the flask 1X with 10 ml of HEPES-buffered RPMI 1640; specific Tcell colonies together with adherent monocytes remained in the flask.Fifty ml of AIM V plus 20 U/ml of human IL-2 (IMMUNEX, Seattle Wash.),and 1 ml (2%, final concentration) pooled human serum were added back tothe flask. After 10-14 days of total incubation time, T cells wereharvested and dead cells were pelleted through Ficoll Hypaque, yieldinga highly enriched population (95-98%) of viable CD4⁺, Ag-specific Tcells. The T cells were confirmed to be specific for TT830 peptide asshown in FIG. 3. Large quantities of monocytes were purified fromleukophoresis packs using the cold aggregation method (Mentzer S. J. etal. (1986) Cell. Immunol. 101:132) which resulted in 80-90% purity. Bothmonocytes and T cells were frozen in aliquots for future use and wereshown to function normally after being thawed.

Ag Presentation Assay

In proliferation assays, T cells (5×10⁴), irradiated monocytes (3000rad, 10⁵/well), and various concentrations of peptide TT830 fusionprotein Fab22-TT830 were incubated together in a final volume of 200μl/well in flat-bottom 96-well tissue culture plates for 2 days. 10 μl(1 μCi/well) ³H-thymidine was then added to each well. After incubatingovernight, plates were harvested and counted in a liquid scintillationcounter. T cell proliferation was expressed as the mean counts/min (CPM)of three replicates ±SD. Background CPM (T cells and monocytes withoutAg) was subtracted from all the data points. Experiments with APL weredone according to similar protocols reported by Sette et al. (DeMagistris (1992) Cell 68:625). Briefly, for inhibition assays,irradiated monocytes were treated with various concentrations of TT 833Sor Fab22-TT833S overnight. 20 nM TT830 and T cells were then added.After a further 2 days incubation, T cell proliferation was measured asdescribed above. In “pre-pulsing” experiments, irradiated monocytes werepulsed with 20 nM TT830 4 h prior to the addition of 10 μM TT 833S or0.1 μM Fab22-TT833S. After overnight incubation, T cells were thenadded. After a further 2 days incubation, T cells were stimulated withirradiated monocytes and TT833S or Fab22-TT833S for 1 day, recoveredafter centrifugation over Ficoll Hypaque, and restimulated withmonocytes and various concentrations of TT830 for 2 days. T cellproliferation was then measured by the incorporation of ³H-thymidine andthe average CPM of three replicates was plotted. In some cases, thepercentage of inhibition was calculated by the formula: %inhibition=(CPM_(no inhibitor)−CPM_(inhibitor))/CPM_(no inhibitor)×100.All experiments were repeated at least three times.

Staining and Flow Cytometry

Staining procedures were adapted from those previously described(Gosselin E. J. et al. (1990) J. Immunol. 144-1817-22). Briefly, toindividual wells of a 96-well plate at 4° C., 30 μl of RPMI+1 mg/ml BSAcontaining one of the proteins Fab22-TT830, Fab22-TT833S, or the BsAbMDXH210 at varying concentrations. After 1 h incubation at 4° C., plateswere centrifuged, the supernatants discarded, and the cells washed threetimes with PBS/BSA at 4° C. Cells were then incubated for 1 h with 40 μl/well of FITC-labeled F(ab′)₂ goat anti-human IgG (JACKSONIMMUNORESEARCH LABORATORIES, INC. West Grove, Pa.) followed by threewashes with PBS/BSA and resuspended in PBS/BSA containing 1%paraformaldehyde (KODAK, Rochester, N.Y.). Cells were then examined byFACScan (BECTON DICKINSON & CO., Mountain View, Calif.), and meanfluorescence intensity (MFI) was measured.

Cytokine Measurement

Supernatants were collected from the 96-well plates of Ag presentationassays after 2 days stimulation and frozen until used. The levels ofIFN-γ and IL-4 from these samples were measured by specific ELISA. Abpairs for the IFN-γ and IL-4-specific ELISA were purchased fromPHARMINGEN (San Diego, Calif.). ELISA assays were performed according tothe protocol provided by the manufacturer.

Generation of H22-TT Peptide Fusion Proteins

In order to generate fusion proteins Fab22-TT830 and Fab22-TT833S,synthetic oligonucleotides encoding each peptide were separatelyengineered into the hinge region in the heavy chain of humanizedanti-FcγRI mAb 22 (H22) according to the method set forth below.

Expression and Cloning Vectors

mAb 22 has been humanized by grafting its CDR regions into a human IgG1framework (see above and Graziano R. F. et al. (1995) J. Immunol.155:4996-5002). The expression vector for the genomic clone of the heavychain (pSVgpt) of H22 was modified to allow incorporation of the codingsequence for other molecules, in this case, the TT peptides. The BamHIfragment of this vector containing CHI, hinge, and newly engineered XhoIand NotI cloning sites (see FIG. 2) was inserted into the BaniHI site ofpUC19 to generate the vector pUC19/H22CH1(X+N). This vector was used toclone oligonucleotide sequences encoding TT peptides, as describedbelow.

The oligonucleotide sequences encoding the tetanus toxin (TT) peptideswere designed to have a XhoI site on the N-terminus and a NotI site onthe C-terminus of the coding region (FIG. 24A). These oligonucleotideswere synthesized and purified by GENOSYS Biotechnologies (The Woodlands,Tex.). The synthetic oligonucleotides were then annealed and ligatedinto the cloning vector pUC19/H22CH1(X+N). Clones which had incorporatedthe coding sequences for TT peptides were screened by restrictionmapping. The BamHI fragment containing CHI, hinge, and TT830 or TT833Swas then cut out of pUC19 and inserted into the expression vector whichalready contained VH. The final expression construct of H22 heavy chainfused with TT peptides is shown in FIG. 24B.

Expression of the H22-TT fusion proteins

The murine myeloma NSO (ECACC 85110503) is a non-Ig synthesizing lineand was used for expression of the H22-TT fusion proteins. First, NSOcells were transfected with the pSVhyg vector containing the H22 lightchain coding sequence. The H22 light chain expressing NSO cells werethen transfected with the expression vector construct containing the H22H-chain Fd sequence fused in frame to the TT coding sequences (FIG.24B). A BioRad Gene Pulser electroporation apparatus was used to carryout the transfection employing 200 v and 960 μFarad. One or two daysafter transfection, mycophenolic acid (0.8 μg/ml; SIGMA) and xanthine(2.5 μg/ml; SIGMA) were added to the media to select transfectants whichhad successfully taken up the expression vectors. Individual colonieswere isolated based on the binding activity of the culture supernatantsto FcγRI on U937 cells as demonstrated by flow cytometry. The positivecolonies were subcloned by limiting dilution.

Purification of the Fab22-TT fusion proteins

Clone pW5 expressing the Fab22-TT830 fusion protein and clone pM4expressing the Fab22-TT833S fusion protein were expanded in rollerbottle cultures. The supernatants were clarified and concentrated. Smallscale purification was performed by affinity chromatography on ananti-H22 affinity column. SDS-PAGE analysis of a 5-10% acrylamidegradient gel under non-reducing conditions showed that fusion proteinswere >90% pure and had a molecular weight of 50 kDa as expected. Proteinconcentration was determined by absorbance at 280 nm using theextinction coefficient of IgG Fab′=1.53.

Results

H22Fd-TT Fusion Proteins Bind to U93 7 Cells

The ability of the H22 fusion proteins, Fab22-TT830 and Fab22-TT833S, tobind to FcγRI was examined first. A previously described bispecific Ab,MDXH210, which contains the same FcγRI-binding component (Fab′ ofhumanized mAb 22) (Valone F. H. et al. (1995) J. Clin. Oncol.13:2281-92), was used as apositive control. Binding of fusion proteinsand MDXH210 to U937 cells, which constitutively express FcγRI, wasmeasured by staining with FITC-labeled goat Ab specific for human IgGand flow cytometry. As indicated in FIGS. 24A and 24B, fusion proteinsFab22-TT830 and Fab22-TT833S bound to U937 cells in a dose-dependentmanner similar to MDXH210. The binding of fusion proteins was completelyblocked by murine anti-human FcγRI mAb 22 F(ab′)₂, demonstrating thespecificity of fusion proteins for FcγRI.

H22Fd-TT Fusion Protein Enhances Presentation of TT Peptide by 100-1000fold.

The fusion protein, Fab22-TT830 was used in Ag presentation assays todetermine whether the Th epitope, TT830, when expressed in the constantregion of H22, could be effectively presented by monocytes to autologousT cells. As shown in FIG. 26, about 1,000-fold less Fab22-TT830 wasrequired than TT830 peptide alone to achieve the same level of T cellproliferation. In addition, FIG. 26 shows that the presentation ofFab22-TT830 was about 10,000-fold more efficient than the presentationof the intact TT, suggesting that the enhanced presentation ofFab22-TT830 did not merely result from higher molecular weight norincreased stability of Fab22-TT830 as opposed to TT830 peptide. Anotherantigenic TT epitope, TT947, failed to stimulate the T cells, confirmingthat the T cells were specific for TT830 peptide. These results thusprovide clear evidence that Th epitopes expressed in the constant regionof H22 can be effectively and specifically presented.

Blockade of FcγRI on Monocytes Abrogates the Enhancement of AgPresentation by the H22Fd-TT Fusion Protein

To directly determine whether the enhancement of peptide presentationthrough the use of the fusion protein is FcγRI-mediated, binding ofFab22-TT830 to FcγRI on Ag-presenting monocytes was blocked by treatingmonocytes with mAb 22 F(ab′)₂ for 1 h prior to the addition ofFab22-TT830 or TT830 peptides. Enhancement of peptide presentation bythe fusion protein was abrogated by mAb 22 F(ab′)₂, whereas presentationof TT830 was unaffected (FIG. 27). The fact that the binding of mAb 22F(ab′)₂ to FcγRI did not lead to an enhancement of the presentation offree peptides implies that binding of mAb 22 to FcγRI alone did notalter the functional state of monocytes in a way that enhanced Agpresentation. Therefore, linkage of the peptide to anti-FcγRI Ab 22appears to be necessary for the observed enhancing effects on Agpresentation, suggesting that the enhanced presentation is probably aresult of efficient Ag capture through FcγRI.

Enhancement of Peptide Presentation by H22 is not Affected by thePresence of Human IgG

Under physiological conditions, the ligand-binding domain of FcγRI issaturated by IgG which blocks efficient targeting of AgAb to thisreceptor. A unique advantage for using derivatives of mAb 22 to triggerFcγRI function is that Ab 22 binds to an epitope outside the ligandbinding domain. Therefore, functions triggered by mAb 22, such as ADCC,phagocytosis and Ag presentation are not inhibited by physiologicallevels of IgG (Gosselin E. J., supra, Guyre P. M. (1989) J. Immunol.143-1650-55). Similarly, the enhanced presentation of the TT830 peptideusing the fusion protein Fab22-TT830 was not inhibited by IgG (FIG. 28),suggesting that H22-based fusion proteins is also an effective way totarget peptide Ags to FcγRI in vivo.

IFN-γ and IL-4 Production is Increased Following H22Fd-TT FusionProtein-enhanced Ag Presentation

Upon activation, T cells not only undergo clonal expansion throughproliferation, but also produce cytokines such as IFN-γ and IL-4 toexert their effector function of B cell differentiation and monocyteactivation (Paul W. E. and Seder, R. A. (1994) Cell 76:241-251).Therefore, the production of IFN-y and IL-4 following H22 fusionprotein-enhanced Ag presentation was examined. As shown in FIGS. 28A and28B, both IFN-γ and IL-4 production levels were enhanced by Fab22-TT830,especially at suboptimal Ag concentrations. However, in theseexperiments, the enhancement for cytokine production (about 20-fold) wasless than that for T cell proliferation (about 600-fold).

Thus, Th epitopes expressed in the constant region of anti-FcγRI mAb H22can be effectively processed and presented by human monocytes, leadingto enhanced T cell activation and cytokine production.

Presentation of APL, TT833S and Fab22-TT833S Fails to Stimulate T CellProliferation

Peptides containing one or two amino acid changes from native T cellepitopes, termed Altered Peptide Ligands (APL) by Allen and coworkers,have been shown to be agonists, partial agonists, or antagonists for Tcell activation (Sette et al. (1994) Ann. Rev. Immunol. 12:413 andEvavold et al. (1993) Immunol. Today 14:602). Recognition of APL byspecific T cells through TCR in some cases triggered partial signaltransduction and resulted in (i) inhibition of T cell stimulation bysuperantigen (Evavlold et al. (1994) Proc. Natl. Acad. Sci. U.S.A.91:2300), T cell anergy (Sloan-Lancaster et al. (1993) Nature 363:156and Sloan-Lancaster et al. (1994) J. Exp. Med. 185:1195), or (iii)modulation of Th1/Th2 differentiation (Nicholson et al. (1995) Immunity3:397; Pfeiffer et al. (1995) J. Exp. Med. 181:1569; and Windhagen etal. (1995) Immunity 2:373). Partial agonists have been shown tostimulate some T cell functions such as IL-4 production by T cells, butnot others such as T cell proliferation (Evabold et al. (1991) Science252:1308). Partial agonists also can induce anergy. Certain APL do nottrigger any detectable signaling events upon interaction with TCR, butcan function as TCR antagonists to inhibit T cell proliferation inresponse to wild-type peptide antigen and are thus called TCRantagonists (De Magistris et al. (1992) Cell 68:625 and Ruppert et al.(1993) Proc. Natl. Acad. Sci. USA 90:2671.

This example demonstrates that the peptide TT833S, an antagonist peptidefor T cell epitope TT830 of tetanus toxin and Fab22-TT833S fail tostimulate T cell proliferation. As shown in FIG. 30, even at doses ashigh as 100 μM for TT833S and 1 μM for Fab22-TT833S, no significantproliferation of TT830-specific T cells was observed. This indicatesthat changing lysine to serine at position 833 of the TT830 peptideeliminated T cell reactivity of this T cell epitope. Additionally, whenpeptides TT830 and TT833S were simultaneously presented toTT830-specific T cells, T cell proliferation in response to TT830 wasinhibited by TT833S in a dose-dependent fashion, showing that TT833S canfunction as an antagonist for TT830-specific T cells (FIG. 31).

Fab22-TT833S is at Least 100 Times More Effective than TT833S inInhibiting T Cell Activation

This example compares the relative efficiency of TT833S and Fab22-TT833Sin inhibiting T cell proliferation in response to TT830. As shown inFIG. 32, Fab22-TT833S was about 100 times more effective than TT833S ininhibiting TT83 0-stimulated T cell proliferation. This suggests thatthe APL, TT833S, when expressed in the constant region of mAb H22, canbe correctly and effectively presented by APC. The increasedantagonistic efficacy of fision protein Fab22-TT833S on T cellproliferation probably reflects more efficient Ag capture mediated byFcγRI as compared to free peptides.

Inhibition of T Cell Activation is Mediated by Competition for T CellReceptor Binding Rather than for MHC Class II Binding.

The antagonist effects of APL TT833S and fusion protein Fab22-TT833Smight be through competition at the level of MHC-binding or TCR-binding,or both. To gain insight into the mechanisms involved, “pre-pulsing”experiments, first described by Sette arid co-workers (DeMagistris, M.T. et al. (1992) Cell 68:625-634), were performed. This experimentalsetting allows agonist (TT830) to bind to MHC Class II in the absence ofcompetition from the inhibitor (TT833S) and thus, only TCR antagonistsbut not pure MHC blockers would be effective in inhibitingagonist-stimulated T cell proliferation. Ag-presenting monocytes werepulsed with suboptimal (20 nM) TT830 for 4 h to allow TT830 to bind withMHC Class II in the absence of competition from TT833S. The APL, TT833Sor Fab22-TT833S, was then incubated with the pre-pulsed monocytes for anadditional 16 h. Responding T cells were added and their proliferationwas measured as described above. Even under such conditions where MHCblockade plays a minimal role, T cell proliferation was still inhibited(FIG. 33). Thus, the inhibition appears to be a result of competitionfor T cell receptor rather than for MHC Class II binding.

Presentation of TT833S and Fab22-TT833S Fails to Stimulate theProduction of IL-4 and IFN-y by T Cells

In some circumstances, APL can stimulate T cell cytokine production butnot proliferation (Evavold B. D. and Allen P. M. (1991) Science252:1308-1310). To determine if presentation of TT833S simulates theproduction of cytokines, the level of IL-4 and IFN-γ in supernatantsobtained from Ag presentation assays was determined. As shown in FIG.34, both TT833S and Fab22-TT833S were ineffective in stimulating IFN-γand IL-4 production by T cells.

Presentation of TT833S and Fab22-TT833S does not Lead to T Cell Anergy

Allen and co-workers reported that interaction of TCR with some APL-MHCClass II complexes led to T cell anergy (Sloan-Lancaster J. et al.(1993) Nature 363:156-159, Sloan-Lancaster J. et al. (1994) J. Exp. Med.180:1195-1205). An experimental scheme similar to theirs was used todetermine whether presentation of TT833S could also cause T cell anergy.As shown in FIG. 35, when T cells were recovered after incubation withAPC and TT833S or Fab22-TT833S for 1 day, 2 days, or 4 days, theyresponded to subsequent antigenic challenge as well as T cells which hadbeen incubated with APC alone. Therefore, the antagonist, presented viathe use of either peptide alone or Fab22-TT833S, did not cause T cellanergy. Furthermore, the same percentage of viable T cells (about 50%)was recovered from cultures with no peptides, TT833S or Fab22-TT833S,suggesting that presentation of TT833S also did not increase T celldeath.

The observation that immunogenicity is increased by about 1000 fold bytargeting antigenic peptides to FcγRI using an anti-FcγRI mAb 22-basedfusion protein indicates that such fusion proteins will be useful forpeptide-based vaccines for, e.g., infectious diseases and cancer.Engineering peptides into the constant domains of human mAb that arespecific for particular APC surface molecules represent a generalapproach to increase the antigenic potency for peptide-based vaccines.

Moreover, the observation that the FcγRI-targeted antagonistic peptideinhibited proliferation of TT830-specific T cells even when APCs werefirst pulsed with native peptide, a situation comparable to that whichwould be encountered in vivo when attempting to ameliorate an autoimmuneresponse show that targeted presentation of antagonistic peptides can beused as Ag-specific therapies for disorders, e.g., T cell-mediatedautoimmune diseases. APL-based treatment will provide anantigen-specific immunotherapy for T cell mediated autoimmune diseasessuch as rheumatoid arthritis and multiple sclerosis. Furthermore, theuse of a fusion protein having one binding specificity to an FcγRI and apeptide which is a partial agonist of an antigen involved in immunedisorders characterized by excessive immune responses will be useful intreating such immune disorders by inducing antigen-specific anergy.Thus, the invention provides methods for treating various immunologicaldisorders, by providing a method allowing for increased antigenpresentation of antigens, which either stimulate T cells, block T cellproliferation and/or cytokine secretion, or which induce anergy in the Tcells.

Example 8

Functional Single Chain anti-FeγRI-anti-CEA Bispecific Molecules

This example demonstrates that a recombinant bispecific single chainmolecule comprising a humanized anti-FcγRI antibody fused to ananti-carcinoembryonic (anti-CEA) antibody is capable of binding to FcγRIand to CEA.

FIG. 37 is a schematic representation of mammalian expression constructsencoding bispecific single chain molecules (constructs 321 and 323)having one binding specificity for the FcγRI and one binding specificityfor carcinoembryonic antigen (CEA) that were prepared. The amino acidsequence of the bispecific single chain molecule H22-anti-CEA encoded byconstruct 321 (SEQ ID NO: 16) and the nucleic acid encoding this fusionprotein (SEQ ID NO: 15) are shown in FIG. 40. The bispecific singlechain molecule H22-anti-CEA encoded by construct 323 differs from thefusion protein encoded by construct 321 only in that the VH and VLchains of H22 were switched.

A mammalian expression construct encoding a single chain antibody havingone binding specificity for the FcγRI (construct 225) was also prepared.The amino acid sequence of the single chain antibody H22 encoded byconstruct 225 (SEQ ID NO: 14) and the nucleic acid encoding this singlechain antibody (SEQ ID NO: 13) are shown in FIG. 39.

Each of these constructs were cloned into the Hind III and XbaI sites ofpcDNA3 (In Vitrogen), from which expression is driven from the CMVpromoter. Each of these constructs also contain a nucleic acid sequenceencoding a peptide from c-myc and a hexa-histidine peptide, which wereused for purification of the recombinant protein from the cell culture.The c-myc tag corresponds to amino acids 410 to 420 of human c-myc (Evanet al. (1985) Mol. Cell. Biol. 5:3610). The anti-CEA single chainantibody, termed MFE-23, is further described in Casey et al. (1994) J.Immunol. Methods 179:105 and Chester et al. (1994) Lancet 343:455.

The single chain bispecific molecules H22-anti-CEA and the single chainH22 antibody were used in binding assays, performed as follows. ELISAplates are coated with CEA and the blocked with 5% PBA. Supernatants ofthe cells transfected with the constructs encoding the single chainmolecules (transfectomas) were added to the plates, soluble Fcγ RI/IgM-μ(supernatant from COS transfected cells, described above) was added andbinding was detected by incubation of the plates withalkaline-phosphatase (AP) conjugated goat anti-human IgM, developmentwith PNPP, and reading of the plate at 405-650 nm.

The results are presented in FIG. 38. The results indicate that thesingle chain bispecific H22-anti-CEA molecules encoded by constructs 321and 323 bind both Fcγ RI and CEA. On the other hand, the single chainH22 antibody (encoded by construct 225) does not bind both FcγrI andCEA, as expected.

Example 9

Targeting CD64 (FcγRI) to Induce and/or Enhance Antigen Processing andPresentation

In order to determine whether directing a normally non-immunogenicantigen (e.g., a self antigen) to human CD64 (FcγRI) can overcomeimmunologic non-responsiveness in vivo, several multimer complexescontaining such antigens linked to F(ab′)2 antibody fragments directedto Fc receptors on antigen presenting cells were prepared and tested asdescribed below. In particular, mice transgenic for human CD64, as wellas their nontransgenic littermates, were immunized with the F(ab′)2fragment of the murine anti-30 human CD64 mAb, M22 (produced by thehybridoma having ATCC Deposit No. HB-12147). The mice were bled sevendays after receiving their fourth biweekly immunization. Upon analysisby ELISA, three of the six transgenic mice, but none of the sixnon-transgenic littermates, exhibited significant anti-M22 Id specificantibody titer. This antisera was M22 specific and did not react withother murine antibodies.

To determine whether multimer complexes of the M22 Fab′ would inducemore efficient internalization of antigen and, thus, enhance antigenpresentation and a stronger anti-M22 Id immune response, multimers ofthe M22 Fab′ molecule were synthesized both chemically and geneticallyand tested. These multimers comprise antigens which can be chemicallylinked to 22 F(ab′)2 (or other Fc receptor binding agents) andadditional 22 Fab′ molecules. The final multimer consists of severalmolecular species that contain, e.g., multiple 22 Fab′ arms (e.g., 2 ormore) and one or more antigen molecules. These species may be purifiedby size exclusion or affinity chromatography if desired. FIG. 41 shows aschematic representation for the development of such chemically linkedmultimeric target antigens.

Chemically synthesized multimers were prepared by incubating F(ab′)2 ofM22 with a 20 molar excess of succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) at roomtemperature for 2 hours. Free SMCC was removed from the resultingcomplex, F(ab′)2-SMCC, by G-25 chromatography. Antigens with free thiol(SH) groups were added to the M22 F(ab′)2-SMCC in a 1:1 molar ratio. Theresulting mixture was incubated overnight at room temperature. Thereaction was stopped by adding an excess of iodoacetamide and theresulting conjugate was purified by size exclusion chromatography.

M22 F(ab )2+-antigen multimers enhance/induce an immune response

FIG. 42A shows a non-reducing SDS-PAGE gel containing purified M22F(ab′)2 (lane 1) and the chemically-linked M22 Fab′multimer (lane 2).The multimer consisted of several molecular species representingF(ab′)3, F(ab′)4, F(ab′)5, F(ab′)6, and higher molecular weightmolecules.

To determine the ability of M22 F(ab′)2, compared to the multimer, M22F(ab′)3⁺, to mediate internalization of human CD64, variousconcentrations of either M22 F(ab′)2 or M22 F(ab′)3⁺ were incubated fortwo hours with macrophages isolated from human CD64-transgenic mice.human CD64 surface expression was detected by the FITC-labeledanti-human CD64 mAb M32.2 (produced by the hybridoma having ATCC DepositNo. 9469) which binds to a site on human CD64 which is distinct from theM22 epitope. As shown in FIG. 42B, incubation with the M22 F(ab′)2 didnot result in significant reduction of human CD64 from the surface ofthe macrophages. However, incubation with the F(ab′)3⁺ at 37° C. led toup to 50% reduction in human CD64 expression in a dose-dependentfashion. Incubation with the F(ab′)3⁺ at 4° C. did not lead to areduction in human CD64 expression, demonstrating thetemperature-dependence of this phenomenon.

Further experiments were performed to determine if modulation wouldoccur in vivo. Human-CD64 transgenic mice were injected with either M22F(ab′)2 or M22 F(ab′)3⁺ and bled one hour later. Surface expression ofhuman CD64 was significantly reduced on circulating monocytes from theM22 F(ab′)3⁺, but not the M22 F(ab′)2 immunized animals. It washypothesized that the reduction of surface human CD64 expressionrepresented internalization of the receptor along with the F(ab′)3⁺multimer.

The ability of human CD64-transgenic and nontransgenic littermates togenerate an anti-M22 idiotype (“Id”) response after three immunizationswith the M22 F(ab′)3⁺ multimer was next studied. The data shown in FIG.43A demonstrates that high titers of M22-specific antibody weregenerated in all of the human CD64-transgenic mice (n=12) immunizedthree times (25 μg/immunization) with the multimer, but no M22-specificantibody was generated in any of the nontransgenic littermates immunizedsimilarly (n=10). The immune response in human CD64-transgenics that waselicited by the M22 F(ab′)3⁺ multimer was signficantly better than thatelicited by the M22 F(ab′)2. All of the multimer-immunized miceresponded and fewer immunizations were required (three versus four) togenerate an immune response immunizing with the multimer as compared tothe F(ab′)2. These results demonstrate that the Fab′ multimer complexeslead to better internalization of an antigen via human CD64, resultingin an enhanced antigen-specific immune response. In fact, the data shownin FIG. 43B demonstrates that immunizing human CD64-transgenic mice withas little as 0.25 μg of the multimer still leads to a detectable immuneresponse in most of the mice.

It is difficult to observe a potent immune response to manytumor-associated antigens since the immune system is often tolerant tothese antigens. In order to determine whether directing such a normallynon-immunogenic antigen to human CD64 resulted in an immune response tothis antigen, a model antigen, the Fab′ fragment of the murinemonoclonal antibody, 520C9, was coupled to the M22 multimer.

After immunization of either human CD64 transgenic or theirnontransgenic littermates, both anti-520C9 Id titers and anti-M22 Idtiters were assessed. As shown in FIG. 44A, seven of eight immunizedhuman CD64 transgenic mice developed both strong anti-520C9 and anti-M22titers. None of the seven immunized nontrangenic mice developedmeasurable titers to either 520C9 or to M22. These data demonstrate thatby coupling a normally non-immunogenic protein to human CD64 so that itis internalized by human CD64⁺ antigen presenting cells leads to apotent immune response to the antigen. Several groups have shown thatdeveloping immunity specific for the idiotype expressed by surface Ig onB cell lymphoma cells can lead to potent tumor cell-specific immunity.The example demonstrates that a specific anti-520C9 Id response can bereadily elicited by directing the 520C9 idiotype to human CD64.

The quality of the immune response to a given antigen, particularly atumor-associated or viral antigen, can be as important as the quantityof the response. T helper cells have been divided into Th1 and Th2depending on the type of cytokines they secrete and also the type ofhelp that the provide to B cells when stimulated. Th1 cells secrete IL-2and IFN-γ when stimulated and usually stimulate B cells to secreteantibody of the IgG2a isotype. Th2 cells secrete IL-4 and IL-5, whenstimulated, and usually stimulate B cells to secrete antibody of theIgG1 or IgE isotype. In general, Th1 cells are believed to stimulate thecellular arm of the immune response whereas Th2 cells stimulate thehumoral arm. The titer of 520C9-specific antibody, of either the IgG 1or the IgG2a isotype, was examined using isotype-specific developingreagents after having immunized human CD64-transgenic mice with 520C9coupled to the M22 multimer. The data shown in FIG. 44B demonstratesthat both a strong IgG1 as well as a strong IgG2a Id-specific titer waselicited, demonstrating the ability of this method of immunization toactivate both a Th1 and a Th2 response.

H22sFv-H22sFv-32.2sFv-antigen Multimer Induces Immune Response

FIG. 45(a) shows the map of a vector encoding a genetically-linkedmultimeric, targeted antigen. This vector encodes for a protein with twosFv regions from humanized anti- FcγRI antibody, H22 (produced by thehybridoma having ATCC Deposit No. CRL 11177), and one sFv region fromantibody M32.2, all linked together to an antigen.

The resulting fusion protein, H22sFv-H22sFv-32.2sFv-antigen (shown inFIG. 45(b)), is capable of binding to FcγRI at multiple sites, andthereby induce internalization via aggregation of the receptor.

The H22sFv-H22sFv-32.2sFv-antigen multimer was made using murine gp75(Trp-1 melanoma antigen) as an antigen for model studies in transgenicmice. It was found that this fusion protein binds FcγRI efficiently andinduces internalization of the receptor. As shown in FIG. 46,chemically-linked M22FabxM22FabxM32Fab and genetically-linkedH22sFv-H22sFv-32.2sFv-antigen each induced internalization of FcγRI. Thestudy was performed by addition of samples to IFN-γ-treated U-937 cellsfor 2 hours at 37° C. FcγRI expression was then measured by stainingcells with Human IgG1-FITC for 1 hour at 4° C., and samples wereanalyzed by FACscan. Percent (%) modulation was calculated by theformula: [1-(MFI of sample/MFI of control)]×100%, wherein MFI is meanfluorescence intensity.

H22sFv2-antigen Binds FcγRI with Affinity Approximately Equal toH22Fab2-antizen

FIG. 47(a) shows a map of a vector encoding a fusion protein made up oftwo H22 sFv regions genetically linked together to an antigen. Theresulting fusion protein, H22sFv-H22sFv-antigen (shown in FIG. 47(b)),is capable of binding to two FcγRI molecules. This fusion protein shouldhave greater affinity for FcγRI than fusion proteins consisting of justone H22 sFv. The ability of this multimer to bind to bind two FcγRImolecules may also induce greater internalization of the receptor.

To test this hypotheses, (i.e., whether the H22sFv-H22sFv-antigen fusionbinds to FcγRI with greater affinity than a single H22sFv-antigenfusion), a competition experiment was performed. U937 cells, previouslytreated with IFN-γ to enhance expression of FcγRI, were stained with anantibody H22 and phycoerythrin conjugate. As shown in FIG. 48, theconjugate was inhibited by various concentrations of H22 Fab,H22sFv2-EGF fusion protein, or H22 Fab2. As also shown in FIG. 48,H22sFv2-EGF binds FcγRI with an affinity approximately equal to that ofH22Fab2. It has been previously shown that H22 Fab′ and H22 sFv havesimilar affinity for FcγRI (Goldstein et al. (1997) J. Immunol.).

H22sFv-H22sFv-H22sFv-CEA Induces Internalization of FcγRI

FIG. 49(a) shows a map of a vector which encodes a multimer fusionprotein containing three-H22 sFv fragments linked to an antigen. Thefusion protein, H22sFv-H22sFv-H22sFv-CEA (shown in FIG. 49(b)), containsthe tumor antigen CEA linked with the 3 H22sFvs. The fusion proteinshould bind FcγRI with high affinity (due to the trivalent binding ofthree sFv molecules), and should also lead to FcγRI internalization byaggregation of three FcγRI molecules.

The ability of the genetically-linked H22sFv-H22sFv-H22sFv-CEA fusion toinduce internalization of FcγRI was tested as previously described, andthe results are shown in FIG. 50. Importantly, the H22Fab-CEA did notmediate internalization at similar concentrations, likely because itbinds monovalently to FcγRI. The example was performed by addition ofsamples to IFN-γ-treated U-937 cells and incubating for either one houror overnight at 37° C. FcγRI expression was then measured by stainingcells with M32.2-FITC for one hour at 4° C., and samples were analyzedby FACscan.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

16 24 base pairs nucleic acid single linear cDNA CDS 1..24 1 ACT CAC ACATGC CCA CCG TGC CCA 24 Thr His Thr Cys Pro Pro Cys Pro 1 5 27 base pairsnucleic acid single linear cDNA CDS 1..19 2 ACT CAC ACA TGC CCA CCG TGAGGATCC 27 Thr His Thr Cys Pro Pro 1 5 42 base pairs nucleic acidsingle linear cDNA CDS 1..34 3 ACT CAC ACA TGC TCG AGC CTT CAC GGC GGCCGC T GAGGATCC 42 Thr His Thr Cys Ser Ser Leu His Gly Gly Arg 1 5 10 300amino acids amino acid linear peptide internal 4 Glu Val Gln Leu Val GluSer Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu SerCys Ser Ser Ser Gly Phe Ile Phe Ser Asp Asn 20 25 30 Tyr Met Tyr Trp ValArg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser AspGly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe ThrIle Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met AspSer Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys 85 90 95 Ala Arg Gly TyrTyr Arg Tyr Glu Gly Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr ProVal Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe ProLeu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 LeuGly Cys Leu Val Lys Asp Tyr Phe Pro Glu Arg Val Thr Val Ser 145 150 155160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn HisLys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys SerCys Asp 210 215 220 Lys Thr His Thr Cys Ser Thr Thr Ser Thr Thr Gly ThrSer His Leu 225 230 235 240 Val Lys Cys Ala Glu Lys Glu Lys Thr Phe CysVal Asn Gly Gly Glu 245 250 255 Cys Phe Met Val Lys Asp Leu Ser Asn ProSer Arg Tyr Leu Cys Lys 260 265 270 Cys Pro Asn Glu Phe Thr Gly Asp ArgCys Gln Asn Tyr Val Met Ala 275 280 285 Ser Phe Tyr Lys Ala Glu Glu LeuTyr Gln Lys Arg 290 295 300 14 amino acids amino acid linear peptideinternal 5 Gln Arg Leu Gly Asn Gln Trp Ala Val Gly His Leu Met Gly 1 510 15 amino acids amino acid linear peptide internal 6 Gln Tyr Ile LysAla Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu 1 5 10 15 54 base pairsnucleic acid single linear cDNA 7 TCGAGCCAGT ACATCAAGGC GAATTCCAAGTTCATCGGCA TCACCGAGCT CTGA 54 53 base pairs nucleic acid single linearcDNA 8 CGGTCATGTA GTTCCGCTTA AGGTTCAAGT AGCCGTAGTG GCTCGAGACT CCG 53 15amino acids amino acid linear peptide internal 9 Gln Tyr Ile Ser Ala AsnSer Lys Phe Ile Gly Ile Thr Glu Leu 1 5 10 15 54 base pairs nucleic acidsingle linear cDNA 10 TCGAGCCAGT ACATCAGCGC GAATTCCAAG TTCATCGGCATCACCGAGCT CTGA 54 53 base pairs nucleic acid single linear cDNA 11CGGTCATGTA GTCGCGCTTA AGGTTCAAGT AGCCGTAGTG GCTCGAGACT CCG 53 21 aminoacids amino acid linear peptide internal 12 Phe Asn Asn Phe Thr Val SerPhe Trp Leu Arg Val Pro Lys Val Ser 1 5 10 15 Ala Ser His Leu Glu 20 913base pairs nucleic acid single linear cDNA CDS 11..911 13 AAGCTTCACC ATGGGA TGG AGC TGT ATC ATC CTC TTC TTG GTG GCC ACA 49 Met Gly Trp Ser CysIle Ile Leu Phe Leu Val Ala Thr 1 5 10 GCT ACC GGT GTC CAC TCC GAT ATCCAA CTG GTG GAG AGC GGT GGA GGT 97 Ala Thr Gly Val His Ser Asp Ile GlnLeu Val Glu Ser Gly Gly Gly 15 20 25 GTT GTG CAA CCT GGC CGG TCC CTG CGCCTG TCC TGC TCC TCG TCT GGC 145 Val Val Gln Pro Gly Arg Ser Leu Arg LeuSer Cys Ser Ser Ser Gly 30 35 40 45 TTC AGT TTC AGT GAC AAT TAC ATG TATTGG GTG AGA CAG GCA CCT GGA 193 Phe Ile Phe Ser Asp Asn Tyr Met Tyr TrpVal Arg Gln Ala Pro Gly 50 55 60 AAA GGT CTT GAG TGG GTT GCA ACC ATT AGTGAT GGT GGT AGT TAC ACC 241 Lys Gly Leu Glu Trp Val Ala Thr Ile Ser AspGly Gly Ser Tyr Thr 65 70 75 TAC TAT CCA GAC AGT GTG AAG GGA AGA TTT ACAATA TCG AGA GAC AAC 289 Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr IleSer Arg Asp Asn 80 85 90 AGC AAG AAC ACA TTG TTC CTG CAA ATG GAC AGC CTGAGA CCC GAA GAC 337 Ser Lys Asn Thr Leu Phe Leu Gln Met Asp Ser Leu ArgPro Glu Asp 95 100 105 ACC GGG GTC TAT TTT TGT GCA AGA GGC TAC TAT AGGTAC GAG GGG GCT 385 Thr Gly Val Tyr Phe Cys Ala Arg Gly Tyr Tyr Arg TyrGlu Gly Ala 110 115 120 125 ATG GAC TAC TGG GGC CAA GGG ACC CCG GTC ACCGTG AGC TCA GGA GGT 433 Met Asp Tyr Trp Gly Gln Gly Thr Pro Val Thr ValSer Ser Gly Gly 130 135 140 GGC GGC TCC GGA GGT GGA GGC AGC GGA GGG GGCGGA TCC GAC ATC CAG 481 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly GlySer Asp Ile Gln 145 150 155 CTG ACC CAG AGC CCA AGC AGC CTG AGC GCC AGCGTG GGT GAC AGA GTG 529 Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser ValGly Asp Arg Val 160 165 170 ACC ATC ACC TGT AAG TCC AGT CAA AGT GTT TTATAC AGT TCA AAT CAG 577 Thr Ile Thr Cys Lys Ser Ser Gln Ser Val Leu TyrSer Ser Asn Gln 175 180 185 AAG AAC TAC TTG GCC TGG TAC CAG CAG AAG CCAGGT AAG GCT CCA AAG 625 Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro GlyLys Ala Pro Lys 190 195 200 205 CTG CTG ATC TAC TGG GCA TCC ACT AGG GAATCT GGT GTG CCA AGC AGA 673 Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu SerGly Val Pro Ser Arg 210 215 220 TTC AGC GGT AGC GGT AGC GGT ACC GAC TTCACC TTC ACC ATC AGC AGC 721 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe ThrPhe Thr Ile Ser Ser 225 230 235 CTC CAG CCA GAG GAC ATC GCC ACC TAC TACTGC CAT CAA TAC CTC TCC 769 Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr CysHis Gln Tyr Leu Ser 240 245 250 TCG TGG ACG TTC GGC CAA GGG ACC AAG GTGGAA ATC AAA TCT AGC TGC 817 Ser Trp Thr Phe Gly Gln Gly Thr Lys Val GluIle Lys Ser Ser Cys 255 260 265 TCG AGC GGA GGC GGG GGT AGC GAT ATC GCGGCC GCA GAA CAG AAA CTC 865 Ser Ser Gly Gly Gly Gly Ser Asp Ile Ala AlaAla Glu Gln Lys Leu 270 275 280 285 ATC TCA GAA GAG GAT CTG AAT GGC GCCGCA CAT CAC CAT CAT CAC CAT 913 Ile Ser Glu Glu Asp Leu Asn Gly Ala AlaHis His His His His His 290 295 300 301 amino acids amino acid linearprotein 14 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala ThrGly 1 5 10 15 Val His Ser Asp Ile Gln Leu Val Glu Ser Gly Gly Gly ValVal Gln 20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ser Ser Ser Gly PheIle Phe 35 40 45 Ser Asp Asn Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly LysGly Leu 50 55 60 Glu Trp Val Ala Thr Ile Ser Asp Gly Gly Ser Tyr Thr TyrTyr Pro 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp AsnSer Lys Asn 85 90 95 Thr Leu Phe Leu Gln Met Asp Ser Leu Arg Pro Glu AspThr Gly Val 100 105 110 Tyr Phe Cys Ala Arg Gly Tyr Tyr Arg Tyr Glu GlyAla Met Asp Tyr 115 120 125 Trp Gly Gln Gly Thr Pro Val Thr Val Ser SerGly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly SerAsp Ile Gln Leu Thr Gln 145 150 155 160 Ser Pro Ser Ser Leu Ser Ala SerVal Gly Asp Arg Val Thr Ile Thr 165 170 175 Cys Lys Ser Ser Gln Ser ValLeu Tyr Ser Ser Asn Gln Lys Asn Tyr 180 185 190 Leu Ala Trp Tyr Gln GlnLys Pro Gly Lys Ala Pro Lys Leu Leu Ile 195 200 205 Tyr Trp Ala Ser ThrArg Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 210 215 220 Ser Gly Ser GlyThr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 225 230 235 240 Glu AspIle Ala Thr Tyr Tyr Cys His Gln Tyr Leu Ser Ser Trp Thr 245 250 255 PheGly Gln Gly Thr Lys Val Glu Ile Lys Ser Ser Cys Ser Ser Gly 260 265 270Gly Gly Gly Ser Asp Ile Ala Ala Ala Glu Gln Lys Leu Ile Ser Glu 275 280285 Glu Asp Leu Asn Gly Ala Ala His His His His His His 290 295 300 1679base pairs nucleic acid single linear cDNA CDS 11..1667 15 AAGCTTCACCATG GGA TGG AGC TGT ATC ATC CTC TTC TTG GTG GCC ACA 49 Met Gly Trp SerCys Ile Ile Leu Phe Leu Val Ala Thr 1 5 10 GCT ACC GGT GTC CAC TCC GATATC CAA CTG GTG GAG AGC GGT GGA GGT 97 Ala Thr Gly Val His Ser Asp IleGln Leu Val Glu Ser Gly Gly Gly 15 20 25 GTT GTG CAA CCT GGC CGG TCC CTGCGC CTG TCC TGC TCC TCG TCT GGC 145 Val Val Gln Pro Gly Arg Ser Leu ArgLeu Ser Cys Ser Ser Ser Gly 30 35 40 45 TTC ATT TTC AGT GAC AAT TAC ATGTAT TGG GTG AGA CAG GCA CCT GGA 193 Phe Ile Phe Ser Asp Asn Tyr Met TyrTrp Val Arg Gln Ala Pro Gly 50 55 60 AAA GGT CTT GAG TGG GTT GCA ACC ATTAGT GAT GGT GGT AGT TAC ACC 241 Lys Gly Leu Glu Trp Val Ala Thr Ile SerAsp Gly Gly Ser Tyr Thr 65 70 75 TAC TAT CCA GAC AGT GTG AAG GGA AGA TTTACA ATA TCG AGA GAC AAC 289 Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe ThrIle Ser Arg Asp Asn 80 85 90 AGC AAG AAC ACA TTG TTC CTG CAA ATG GAC AGCCTG AGA CCC GAA GAC 337 Ser Lys Asn Thr Leu Phe Leu Gln Met Asp Ser LeuArg Pro Glu Asp 95 100 105 ACC GGG GTC TAT TTT TGT GCA AGA GGC TAC TATAGG TAC GAG GGG GCT 385 Thr Gly Val Tyr Phe Cys Ala Arg Gly Tyr Tyr ArgTyr Glu Gly Ala 110 115 120 125 ATG GAC TAC TGG GGC CAA GGG ACC CCG GTCACC GTG AGC TCA GGA GGT 433 Met Asp Tyr Trp Gly Gln Gly Thr Pro Val ThrVal Ser Ser Gly Gly 130 135 140 GGC GGC TCC GGA GGT GGA GGC AGC GGA GGGGGC GGA TCC GAC ATC CAG 481 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly GlyGly Ser Asp Ile Gln 145 150 155 CTG ACC CAG AGC CCA AGC AGC CTG AGC GCCAGC GTG GGT GAC AGA GTG 529 Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala SerVal Gly Asp Arg Val 160 165 170 ACC ATC ACC TGT AAG TCC AGT CAA AGT GTTTTA TAC AGT TCA AAT CAG 577 Thr Ile Thr Cys Lys Ser Ser Gln Ser Val LeuTyr Ser Ser Asn Gln 175 180 185 AAG AAC TAC TTG GCC TGG TAC CAG CAG AAGCCA GGT AAG GCT CCA AAG 625 Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys ProGly Lys Ala Pro Lys 190 195 200 205 CTG CTG ATC TAC TGG GCA TCC ACT AGGGAA TCT GGT GTG CCA AGC AGA 673 Leu Leu Ile Tyr Trp Ala Ser Thr Arg GluSer Gly Val Pro Ser Arg 210 215 220 TTC AGC GGT AGC GGT AGC GGT ACC GACTTC ACC TTC ACC ATC AGC AGC 721 Phe Ser Gly Ser Gly Ser Gly Thr Asp PheThr Phe Thr Ile Ser Ser 225 230 235 CTC CAG CCA GAG GAC ATC GCC ACC TACTAC TGC CAT CAA TAC CTC TCC 769 Leu Gln Pro Glu Asp Ile Ala Thr Tyr TyrCys His Gln Tyr Leu Ser 240 245 250 TCG TGG ACG TTC GGC CAA GGG ACC AAGGTG GAA ATC AAA TCT AGC TGC 817 Ser Trp Thr Phe Gly Gln Gly Thr Lys ValGlu Ile Lys Ser Ser Cys 255 260 265 TCG AGC GGA GGC GGG GGT AGC GAT ATCAAA CTG CAG CAG TCT GGG GCA 865 Ser Ser Gly Gly Gly Gly Ser Asp Ile LysLeu Gln Gln Ser Gly Ala 270 275 280 285 GAA CTT GTG AGG TCA GGG ACC TCAGTC AAG TTG TCC TGC ACA GCT TCT 913 Glu Leu Val Arg Ser Gly Thr Ser ValLys Leu Ser Cys Thr Ala Ser 290 295 300 GGC TTC AAC ATT AAA GAC TCC TATATG CAC TGG TTG AGG CAG GGG CCT 961 Gly Phe Asn Ile Lys Asp Ser Tyr MetHis Trp Leu Arg Gln Gly Pro 305 310 315 GAA CAG GGC CTG GAG TGG ATT GGATGG ATT GAT CCT GAG AAT GGT GAT 1009 Glu Gln Gly Leu Glu Trp Ile Gly TrpIle Asp Pro Glu Asn Gly Asp 320 325 330 ACT GAA TAT GCC CCG AAG TTC CAGGGC AAG GCC ACT TTT ACT ACA GAC 1057 Thr Glu Tyr Ala Pro Lys Phe Gln GlyLys Ala Thr Phe Thr Thr Asp 335 340 345 ACA TCC TCC AAC ACA GCC TAC CTGCAG CTG AGC AGC CTG ACA TCT GAG 1105 Thr Ser Ser Asn Thr Ala Tyr Leu GlnLeu Ser Ser Leu Thr Ser Glu 350 355 360 365 GAC ACT GCC GTC TAT TAT TGTAAT GAG GGG ACT CCG ACT GGG CCG TAC 1153 Asp Thr Ala Val Tyr Tyr Cys AsnGlu Gly Thr Pro Thr Gly Pro Tyr 370 375 380 TAC TTT GAC TAC TGG GGC CAAGGG ACC ACG GTC ACC GTC TCC TCA GGT 1201 Tyr Phe Asp Tyr Trp Gly Gln GlyThr Thr Val Thr Val Ser Ser Gly 385 390 395 GGA GGC GGT TCA GGC GGA GGTGGC TCT GGC GGT GGC GGA TCA GAA AAT 1249 Gly Gly Gly Ser Gly Gly Gly GlySer Gly Gly Gly Gly Ser Glu Asn 400 405 410 GTG CTC ACC CAG TCT CCA GCAATC ATG TCT GCA TCT CCA GGG GAG AAG 1297 Val Leu Thr Gln Ser Pro Ala IleMet Ser Ala Ser Pro Gly Glu Lys 415 420 425 GTC ACC ATA ACC TGC AGT GCCAGC TCA AGT GTA AGT TAC ATG CAC TGG 1345 Val Thr Ile Thr Cys Ser Ala SerSer Ser Val Ser Tyr Met His Trp 430 435 440 445 TTC CAG CAG AAG CCA GGCACT TCT CCC AAA CTC TGG ATT TAT AGC ACA 1393 Phe Gln Gln Lys Pro Gly ThrSer Pro Lys Leu Trp Ile Tyr Ser Thr 450 455 460 TCC AAC CTG GCT TCT GGAGTC CCT GCT CGC TTC AGT GGC AGT GGA TCT 1441 Ser Asn Leu Ala Ser Gly ValPro Ala Arg Phe Ser Gly Ser Gly Ser 465 470 475 GGG ACC TCT TAC TCT CTCACA ATC AGC CGA ATG GAG GCT GAA GAT GCT 1489 Gly Thr Ser Tyr Ser Leu ThrIle Ser Arg Met Glu Ala Glu Asp Ala 480 485 490 GCC ACT TAT TAC TGC CAGCAA CGG AGT AGT TAC CCA CTC ACG TTC GGT 1537 Ala Thr Tyr Tyr Cys Gln GlnArg Ser Ser Tyr Pro Leu Thr Phe Gly 495 500 505 GCT GGC ACC AAG CTG GAGCTG AAA CGG GCG GCA GGC TCG AGC GGA GGC 1585 Ala Gly Thr Lys Leu Glu LeuLys Arg Ala Ala Gly Ser Ser Gly Gly 510 515 520 525 GGG GGT AGC GAT ATCGCG GCC GCA GAA CAG AAA CTC ATC TCA GAA GAG 1633 Gly Gly Ser Asp Ile AlaAla Ala Glu Gln Lys Leu Ile Ser Glu Glu 530 535 540 GAT CTG AAT GGC GCCGCA CAT CAC CAT CAT CAC CAT TGATTCTAGA 1679 Asp Leu Asn Gly Ala Ala HisHis His His His His 545 550 553 amino acids amino acid linear protein 16Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 1015 Val His Ser Asp Ile Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 2530 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ser Ser Ser Gly Phe Ile Phe 35 4045 Ser Asp Asn Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 5560 Glu Trp Val Ala Thr Ile Ser Asp Gly Gly Ser Tyr Thr Tyr Tyr Pro 65 7075 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 8590 95 Thr Leu Phe Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val100 105 110 Tyr Phe Cys Ala Arg Gly Tyr Tyr Arg Tyr Glu Gly Ala Met AspTyr 115 120 125 Trp Gly Gln Gly Thr Pro Val Thr Val Ser Ser Gly Gly GlyGly Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile GlnLeu Thr Gln 145 150 155 160 Ser Pro Ser Ser Leu Ser Ala Ser Val Gly AspArg Val Thr Ile Thr 165 170 175 Cys Lys Ser Ser Gln Ser Val Leu Tyr SerSer Asn Gln Lys Asn Tyr 180 185 190 Leu Ala Trp Tyr Gln Gln Lys Pro GlyLys Ala Pro Lys Leu Leu Ile 195 200 205 Tyr Trp Ala Ser Thr Arg Glu SerGly Val Pro Ser Arg Phe Ser Gly 210 215 220 Ser Gly Ser Gly Thr Asp PheThr Phe Thr Ile Ser Ser Leu Gln Pro 225 230 235 240 Glu Asp Ile Ala ThrTyr Tyr Cys His Gln Tyr Leu Ser Ser Trp Thr 245 250 255 Phe Gly Gln GlyThr Lys Val Glu Ile Lys Ser Ser Cys Ser Ser Gly 260 265 270 Gly Gly GlySer Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Val 275 280 285 Arg SerGly Thr Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn 290 295 300 IleLys Asp Ser Tyr Met His Trp Leu Arg Gln Gly Pro Glu Gln Gly 305 310 315320 Leu Glu Trp Ile Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr 325330 335 Ala Pro Lys Phe Gln Gly Lys Ala Thr Phe Thr Thr Asp Thr Ser Ser340 345 350 Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp ThrAla 355 360 365 Val Tyr Tyr Cys Asn Glu Gly Thr Pro Thr Gly Pro Tyr TyrPhe Asp 370 375 380 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser GlyGly Gly Gly 385 390 395 400 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly SerGlu Asn Val Leu Thr 405 410 415 Gln Ser Pro Ala Ile Met Ser Ala Ser ProGly Glu Lys Val Thr Ile 420 425 430 Thr Cys Ser Ala Ser Ser Ser Val SerTyr Met His Trp Phe Gln Gln 435 440 445 Lys Pro Gly Thr Ser Pro Lys LeuTrp Ile Tyr Ser Thr Ser Asn Leu 450 455 460 Ala Ser Gly Val Pro Ala ArgPhe Ser Gly Ser Gly Ser Gly Thr Ser 465 470 475 480 Tyr Ser Leu Thr IleSer Arg Met Glu Ala Glu Asp Ala Ala Thr Tyr 485 490 495 Tyr Cys Gln GlnArg Ser Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr 500 505 510 Lys Leu GluLeu Lys Arg Ala Ala Gly Ser Ser Gly Gly Gly Gly Ser 515 520 525 Asp IleAla Ala Ala Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn 530 535 540 GlyAla Ala His His His His His His 545 550

We claim:
 1. A molecular complex comprising: a) two or more protein orpeptide binding specificities for a component on the surface of anantigen presenting cell; and b) at least one antigen linked to saidbinding specificities, wherein said component mediates internalizationof said molecular complex when bound by said binding specificities. 2.The molecular complex of claim 1, wherein said complex comprises threeor more binding specificities.
 3. The molecular complex of claim 1,wherein at least one of binding specificities binds to an Fc receptor(FcR) on an antigen presenting cell.
 4. The molecular complex of claim1, wherein said binding specificities comprise an antibody or an antigenbinding fragment thereof.
 5. The molecular complex of claim 4, whereinsaid antibody binds to an Fc receptor (FcR).
 6. The molecular complex ofclaim 5, wherein said FcR is an Fcγ Receptor.
 7. The molecular complexof claim 6, wherein said Fcγ Receptor is FcγRI.
 8. The molecular complexof claim 1, wherein one or more of said binding specificities comprisesan antibody selected from H22 (ATCC Deposit No.CRL 11177), M22 (ATCCDeposit No. HB12147), M32.2 (ATCC Deposit No. HB 9469), and antigenbinding fragments thereof.
 9. The molecular complex of claim 1, whereinsaid binding specificities bind to the same epitope of said cell surfacecomponent.
 10. The molecular complex of claim 1, wherein said bindingspecificities bind to different epitopes of said cell surface component.11. The molecular complex of claim 1, wherein said binding specificitiesbind to different cell surface components.
 12. The molecular complex ofclaim 1, wherein said antigen is chemically linked to said bindingspecificities.
 13. The molecular complex of claim 1, wherein saidantigen is recombinantly fused to said binding specificities.
 14. Themolecular complex of claim 1, wherein said antigen is a tumor antigen.15. The molecular complex of claim 13, wherein said tumor antigen isfrom a cancer selected from the group consisting of breast cancer,sarcoma, carcinoma, and ovarian cancer.
 16. The molecular complex ofclaim 1, wherein said antigen is an autoantigen.
 17. The molecularcomplex of claim 1, wherein said antigen is a self antigen.
 18. Themolecular complex of claim 1, wherein said antigen binds to FcγRI. 19.The molecular complex of claim 1, wherein said antigen comprises anantibody selected from H22 (ATCC Deposit No.CRL 11177), M22 (ATCCDeposit No. HB 12147), M32.2 (ATCC Deposit No. HB 9469), and antigenbinding fragments thereof.
 20. The molecular complex of claim 1, whereinsaid antigen comprises antibody 520C9 (ATCC Deposit No. HB 8696) or anantigen binding fragment thereof.